WO2025058988A1 - Method of treating non-immunogenic skin cancers - Google Patents

Abstract

Described herein are methods and compositions including entinostat and one or more of a TLR7/8 agonist, vitamin D analog, or 5-FU for the treatment of a noninflammatory skin cancer and/or basal cell carcinoma (BCC). Specifically, the disclosures provides methods for treating BCC and/or a noninflammatory skin cancer, the methods comprising administering an effective amount of a histone deacetylase (HD AC) inhibitor in combination with an effective amount of one or more of a TLR7/8 agonist, vitamin D analog, or 5-FU, and wherein the HDAC inhibitor comprises entinostat.

Description

METHOD OF TREATING NON-IMMUNOGENIC SKIN CANCERS

CLAIM OF PRIORITY

This application claims the benefit of U.S. Provisional Application Serial No. 63/582,260, filed on September 13, 2023. The entire contents of the foregoing are incorporated herein by reference.

TECHNICAL FIELD

The present disclosure relates to methods of treating non-immunogenic skin cancers.

BACKGROUND

Basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (SCC) are the most common cancers in the United States and in light-skinned populations worldwide^{1, 2}. Both BCC and SCC are derived from keratinocytes and regarded as keratinocyte carcinoma. In the United States, keratinocyte carcinomas are more common than all other cancers combined^{1, 2} and their incidence is on the rise³.

BCC and SCC have one of the highest tumor mutational burden (TMB) among all cancers⁴. The median TMB is 47.3-90 mutations per megabase (muts/Mb) for BCC and 45.2 muts/Mb for SCC^{4, 5}. It is well established that there is a positive correlation between TMB and objective response rate (ORR) to immunotherapy, including anti- PD-1 or anti-PD-Ll therapy⁶. Consistent with this paradigm, metastatic SCC has shown one of the highest response rates with an ORR of 49.2% to anti-PD-1 therapy⁷. However, metastatic BCC has shown an ORR of only 21% to anti-PD-1 therapy⁸. This observation suggests that BCC is not immunogenic despite its high TMB. Therefore, the need still exists to develop novel treatments for BCCs and SCCs.

SUMMARY

Provided herein are methods of treating or preventing basal cell carcinoma in a subject comprising administering to the subject an effective amount of an HD AC inhibitor in combination with an effective amount of one or more of a TLR7/8 agonist, vitamin D analog, or 5-FU. Provided herein are methods of treating of preventing basal cell carcinoma in a subject comprising administering to the subject an effective amount of an HD AC inhibitor, a systemic immunotherapy, and one or more of a TLR7/8 agonist, vitamin D analog, or 5-FU.

Provided herein are methods of treating or preventing non-immunogenic skin cancers comprising administering to the subject an effective amount of an HD AC inhibitor, in combination with an effective amount of a TLR7/8 agonist, vitamin D analog, or 5-FU.

Provided herein are methods of treating or preventing a Foxcl positive tumor comprising administering to the subject an effective amount of an HD AC inhibitor in combination with an effective amount of one or more of a TLR7/8 agonist, vitamin D analog, or 5-FU.

In some embodiments, the Foxcl positive tumor is a tumor that comprises one or more Foxcl positive cells. In some embodiments, the HDAC inhibitor comprises panobinostat, abexinostat, entinostat, mocetinostat, vorinostat, pracinostat, ricolinostat, apicidin, fimepinostat, P300 histone acetylatransferase, tubastatin A, tucidinostat, romidepsin, belinostat, givinostat, or valproic acid. In some embodiments, the HDAC inhibitor comprises entinostat. In some embodiments, the HDAC inhibitor is administered topically or systemically. In some embodiments, the systemic immunotherapy is an immune checkpoint inhibitor. In some embodiments, the systemic immunotherapy is a sonic hedgehog pathway inhibitor selected from vismodegib and sonidegib. In some embodiments, the TLR7/8 agonist is selected from imiquimod, resiquimod, 852-A, vesatolimod, AZD8848, motolimod, and selgantolimod. In some embodiments, the TLR7/8 agonist is imiquimod. In some embodiments, the non-immunogenic skin cancer comprises Extramammary Paget’s disease, chest wall metastasis of breast cancer, malignant melanoma, or adnexal carcinoma. In some embodiments, the vitamin D analog comprises doxercalciferol, paricalcitol, calcitriol, tacalcitol, alfacalcidol, calcijex, calcipotriol, falecalcitriol, vitamin D, dihydroxycholecalciferol, rayaldee, rocaltrol, calcifediol, cholecalciferol, decara, oleovitamin d3, or trionex. In some embodiments, the vitamin D analog comprises calcipotriol. In some embodiments, the HDAC inhibitor is administered separately from the one or more of a TLR7/8 agonist, vitamin D analog, or 5-FU. In some embodiments, the HDAC inhibitor is administered concomitantly or sequentially. In some embodiments, the HD AC inhibitor is administered together with the one or more of a TLR7/8 agonist, vitamin D analog, or 5-FU in the same composition. In some embodiments, the administering comprises administering the composition(s) to a tumor.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1: BCC has low immunogenicity. FIG. 1A: Representative images of hematoxylin and eosin (H&E), CD3 (low magnification), and CD4/CD8 (high magnification) stained human primary SCC and BCC. Note that CD4+ and CD8+ cells are CD3+ T cells. FIGs. IB and C: Quantification of CD4+ T (FIG. IB) and CD8+ T (FIG. 1C) cells as percent DAPI+ cells in the parenchyma of SCC and BCC. Each dot represents a tumor sample (n = 35 in each group, Mann-Whitney U test). FIG. ID: Uniform manifold approximation and projection (UMAP) plot of human SCC and BCC cells. 10,314 (SCC) and 33,112 (BCC) total cells were analyzed from independent donors (n = 3 in SCC, n = 4 in BCC, Treg: regulatory T cell). Bar graph shows the number of cells in each cellular cluster as % total cells (chi-squared test). FIG. IE: Representative images of HLA-I stained SCC and BCC. FIG. IF: Quantification of HLA-Ihigh cancer cells as percent DAPI+ cells in SCC and BCC samples. Each dot represents a tumor sample (n = 35 in each group, Mann-Whitney U test). FIG. 1G: Representative image of HLA-I and CD3 stained early BCC. The arrow points to a rare CD3+ T cell in tumor stroma. FIG. 1H: Heatmap of APM genes expression in normal keratinocyte cell lines (KERTr, HaCaT), SCC cell lines (SCC 12, SCC13), and a BCC cell line (UW-BCC1) from RNA-Seq analysis. FIGs. II and 1J: Representative flow cytometry histogram (FIG. II) and relative fluorescence intensity (RFI) (FIG. 1 J) of HLA-I on the surface of normal keratinocyte, SCC, and BCC cell lines (n = 6 in each group, one-way ANOVA with Dunnett’s multiple comparison test). Bar graphs show mean + SD, scale bars: 100 pm. FIG. IK: Representative images of CD11c stained SCC and BCC. FIG. IL: Quantification of CD1 lc+ dendritic cells (DCs) as percent DAPI+ cells in the parenchyma of SCC and BCC. Each dot represents a tumor sample (n = 35 in each group, Mann-Whitney U test). FIG. IM: Representative images of CD207 stained SCC and BCC. FIG. IN: Quantification of CD207+HLA-II+ Langerhans cells as percent DAPI+ cells in the parenchyma of SCC and BCC. Each dot represents a tumor sample (n = 35 in each group, Mann-Whitney U test). FIG. 10: Representative images of CD45 and CD56 stained SCC and BCC. FIG. IP: Quantification of CD45+CD56+ NK cells as percent DAPI+ cells in the parenchyma of SCC and BCC. Each dot represents a tumor sample (n = 35 in each group, Mann-Whitney U test). FIG. IQ: UMAP plot of T cells in human SCC and BCC. 1307 (SCC) and 433 (BCC) total T cells were analyzed from independent donors (n = 3 in SCC, n = 4 in BCC). Bar graph shows the number of cells in each T cell cluster as % total T cells (chi-squared test). FIG. 1R: Representative images of P-2 microglobulin (P2M) stained SCC and BCC. FIG. IS: Quantification of p2Mhigh cancer cells as percent DAPI+ cells in the parenchyma of SCC and BCC. Each dot represents a tumor sample (n = 35 in each group, Mann- Whitney U test). FIGs. T and U: Representative flow cytometry histogram (FIG. IT) and RFI (FIG. 1U) of P2M on the surface of normal keratinocyte (KERTr, HaCaT), SCC (SCC12, SCC13), and BCC (UWBCC1) cell lines (n = 6 per group, one-way ANOVA with Dunnett’s multiple comparison test). FIG. IV: RFI of HLA-I on the surface of UW-BCC1 cells treated with 100 nM vismodegib versus DMSO vehicle control (n = 4 in each group, Mann-Whitney U test). Bar graphs show mean + SD, scale bars: 100 pm.

FIG. 2: Foxcl downregulates APM in BCC cells. FIG. 2A: Representative immunoblot of IRFl, NLRC5, phospho-NF-kB, total NF-kB, Foxcl, and GAPDH proteins in normal keratinocyte (KERTr, HaCaT), SCC (SCC12, SCC13), and BCC (UW-BCC1) cell lines. FIGs. 2B-D: Quantification of IRFl (FIG. 2B), NLRC5 (FIG. 2C), and Foxcl (FIG. 2D) protein levels in normal keratinocyte, SCC, and BCC cell lines. GAPDH is used as the control housekeeping protein (n = 5 in each group except KERTr for NLRC5 and Foxcl, n = 4 KERTr for NLRC5 and Foxcl, one-way ANOVA with Dunnett’s multiple comparison test). FIGs. 2E and 2F: Representative flow cytometry histogram (FIG. 2E) and RFI (FIG. 2F) of HLA-I on the surface of UW-BCC1 cells infected with lentivirus vector expressing IRFl (LV-IRF1) compared with control vector (LV-Ctrl) (n = 4 in each group, Mann-Whitney U test). FIGs. 2G and 2H: Representative flow cytometry histogram (FIG. 2G) and RFI (FIG. 2H) of HLA-I on the surface of UW-BCC1 cells transfected with Foxcl siRNA construct (siRNA-Foxcl) versus control siRNA (siRNA-Ctrl) (n = 4 in each group, Mann- Whitney U test). FIG. 21: Representative immunoblot of Foxcl, IRFl, and GAPDH proteins in UW-BCC1 cells expressing IRFl versus control lentivirus vector (LV). FIG. 2J: Quantification of Foxcl protein in UW-BCC1 cells expressing IRFl versus control LV. GAPDH is used as the control housekeeping protein (n = 4 in each group, Mann-Whitney U test). FIGs. 2K and 2L: Representative flow cytometry histogram (K) and RFI (L) of IRFl levels in UW-BCC1 cells transfected with siRNA-Foxcl versus siRNA-Ctrl (n = 4 in each group, Mann-Whitney U test). FIGs. 2M-2O) ChlP- qPCR assay on IRFl (FIG. 2M), HLA-A (FIG. 2N), and B2M (FIG. 20) promoter region using an anti-Foxcl antibody. Control IgG ChlP-qPCR results are shown as negative controls (n = 4 in each group, Mann-Whitney U test). FIG. 2P: Diagram depicting Foxcl regulation of IRFl and APM genes. FHRE: forkhead response element, ISRE: Interferon-stimulated response element. Bar graphs show mean + SD. FIG. 2Q Heatmap of APM genes and major regulator of APM genes expression in normal keratinocyte (KERTr, HaCaT), SCC (SCC12, SCC13), and BCC (UW-BCC1) cell lines from RNA-Seq analysis. FIGs. 2R and S: Representative flow cytometry histogram (FIG. 2R) and RFI (FIG. 2S) of IRFl in normal keratinocytes, SCC, and BCC cell lines (n = 6 in each group, one-way ANOVA with Dunnett’s multiple comparison test). FIGs. 2T and 2U: Representative flow cytometry histogram (FIG. 2T) and RFI (FIG. 2U) of IRFl in UW-BCC1 cells expressing LV-IRF1 compared with negative control (LV-Ctrl) (n = 4 in each group, Mann-Whitney U test). FIGs. 2V and 2W: Representative flow cytometry histogram (FIG. 2V) and RFI (FIG. 2W) of P2M on UW-BCC1 cells expressing LV-IRFl compared with LV-Ctrl (n = 4 in each group, Mann-Whitney U test). FIG. 2X: Relative FOXCI mRNA expression in UW-BCC1 cells transfected with Foxcl siRNA construct (siRNA-Foxcl) versus control siRNA (siRNA-Ctrl). GAPDH is used as the control housekeeping gene (n = 4 in each group, Mann-Whitney U test). FIG. 2Y: Representative immunoblot of Foxcl and GAPDH proteins in UW-BCC1 cells transfected with siRNA-Foxcl or siRNA- Ctrl. FIGs. 2Z and 2AA: Representative flow cytometry histogram (FIG. 2Z) and RFI (FIG. 2AA) of P2M on the surface of UWBCC1 cells transfected with siRNA-Foxcl or siRNA-Ctrl (n = 4 in each group, Mann-Whitney U test). FIGs. 2BB-2FF : Relative HLA-A (FIG. 2BB), HLA-B (FIG. 2CC), HLA-C (FIG. 2DD), B2M (FIG. 2EE), and IRF1 (FIG. 2FF) mRNA expression in UW-BCC1 cells transfected with siRNA- Foxcl or siRNA-Ctrl. GAPDH is used as the control housekeeping gene (n = 6 in each group, Mann-Whitney U test). Bar graphs show mean + SD.

FIG. 3: Entinostat overcomes APM downregulation by Foxcl and enables BCC immunotherapy. FIGs. 3 A and 3B: ChlP-qPCR assay on HLA-A (FIG. 3 A) and B2M (FIG. 3B) promoter region using an anti-H3K27ac antibody on UW-BCC1 cells transfected with Foxcl siRNA construct (siRNA Foxcl) versus control siRNA (siRNA-Ctrl). Control IgG ChlP-qPCR results are shown as negative controls (n = 4 in each group, one-way ANOVA with Dunnett’s multiple comparison test). FIG. 3C: Diagrams showing Foxcl recruitment of HD AC-mediated histone deacetylation leading to the suppression of APM gene expression, which can be reversed by HD AC inhibitors. FIGs. 3D and 3E: Representative flow cytometry histogram (FIG. 3D) and RFI (FIG. 3E) of HLA-I on the surface of UW-BCC1 cells treated with 1 pM entinostat or DMSO vehicle control (n = 4 in each group, Mann-Whitney U test). FIGs. 3F and 3G: Representative flow cytometry histogram (FIG. 3F) and RFI (FIG. 3G) of HLA-I on the surface of UW-BCC1 cells transfected with siRNA-Foxcl or siRNA-Ctrl and treated with 1 pM entinostat or DMSO (n = 6 in each group, one-way ANOVA with Dunnett’s multiple comparison test). FIG. 3H: Schematic diagram of topical therapy for primary BCCs that develop spontaneously in Ptchl+/- mice exposed to DMBA and UVB. FIG. 31: Representative image of Foxcl and CK17 stained mouse BCC in Ptchl+/- skin. (J) Representative images of cytokeratin 17 (CK17) and P2M stained mouse BCCs in Ptchl+/- skin treated with entinostat versus DMSO. Yellow arrows point to CK17+ primary BCCs in the skin. FIG. 3K: Quantification of p2Mhigh cancer cells as percent DAPI+ cells in BCCs of Ptchl+/- skin treated with entinostat versus DMSO. Each dot represents a mouse (n = 15 in DMSO and n = 16 in the entinostat group, Mann-Whitney U test). FIG. 3L: Representative images of CK17 stained skin of Ptchl+/- mice treated with vehicle, entinostat, imiquimod, or entinostat plus imiquimod. Yellow arrows point to CK17+ primary BCCs in the skin. FIG. 3M: Quantification of BCCs in Ptchl+/- skin per length of the skin treated with vehicle, entinostat, imiquimod, or entinostat plus imiquimod. Each dot represents a mouse (n = 15 in DMSO, n = 16 in entinostat, n = 9 in imiquimod, and n = 15 in entinostat plus imiquimod group, one-way ANOVA with Dunnett’s multiple comparison test). Bar graphs show mean + SD, scale bars: 100pm. FIG. 3N: ChlP-qPCR assay on IRF1 promoter region using an anti-H3K27ac antibody on UW-BCC1 cells transfected with Foxcl siRNA construct (siRNA-Foxcl) versus control siRNA (siRNA-Ctrl). Control IgG ChlP-qPCR results are shown as negative controls (n = 4 in each group, one-way ANOVA with Dunnett’s multiple comparison test). FIGs. 30 and 3P: Representative flow cytometry histogram (FIG. 30) and RFI (FIG. 3P) of HLA-I on the surface of UW-BCC1 cells treated with 10 nM romidepsin, 1 pM remetinostat, 0.1 pM givinostat, 1 pM vorinostat, 1 pM entinostat, or DMSO vehicle control (n = 4 in each group, one-way ANOVA with Dunnett’s multiple comparison test). FIGs. 3Q and R: Representative flow cytometry histogram (FIG. 3Q) and RFI (FIG. 3R) of P2M on the surface of UWBCC1 cells treated with 1 pM entinostat or DMSO (n = 4 in each group, Mann-Whitney U test). FIG. 3S and 3T: Representative flow cytometry histogram (FIG. 3S) and RFI (FIG. 3T) of IRF1 in UW-BCC1 cells treated with 1 pM entinostat or DMSO (n = 4 in each group, Mann- Whitney U test). FIGs. 3U and 3 V: Representative flow cytometry histogram (FIG. 3U) and RFI (FIG. 3 V) of HLA-II on the surface of UW-BCC1 cells treated with 1 pM entinostat or DMSO (n = 4 in each group, Mann-Whitney U test). FIGs. 3W-3Z: Relative HLA-A (FIG. 3W), HLA-B (FIG. 3X), HLA-C (FIG. 3 Y), and B2M (FIG. 3Z) mRNA expression in UW-BCC1 cells treated with 1 pM entinostat or DMSO. GAPDH is used as the control housekeeping gene (n = 6 in DMSO and n = 5 in the entinostat group, Mann-Whitney U test). FIGs. 3AA and 3BB: Representative flow cytometry histogram (FIG. 3AA) and RFI (FIG. 3BB) of HLA-I on the surface of SCC 12 cells treated with 1 pM entinostat or DMSO (n = 4 in each group, Mann- Whitney U test). FIGs. 3CC and 3DD: Representative flow cytometry histogram (FIG. 3CC) and RFI (FIG. 3DD) of P2M on the surface of SCC12 cells treated with 1 pM entinostat or DMSO (n = 4 in each group, Mann-Whitney U test). FIGs. 3EE and FF: Representative flow cytometry histogram (FIG. 3EE) and RFI (FIG. 3FF) of IRFl in SCC12 cells treated with 1 pM entinostat or DMSO (n = 4 in each group, Mann- Whitney U test). Bar graphs show mean + SD.

FIG. 4: Primary human BCC’s low immunogenicity is linked to high Foxcl and low IRF1 expression. FIG. 4 A: Representative images of IRF1 and HLA-I stained SCC and BCC. FIG. 4B: Quantification of IRF1+ cancer cells as percent DAPI+ cells in the parenchyma of SCC and BCC. Each dot represents a tumor sample (n = 35 in each group, Mann-Whitney U test). FIG. 4C: Representative images of Foxcl and HLA-I stained SCC and BCC. FIG. 4D: Quantification of HLA-I^mcdll,m cancer cells as percent Foxcl- versus Foxcl + DAPI+ cells in the parenchyma of BCC. HLA-I^mcdllim cells were defined to show an HLA-I signal higher than 50% of the HLA-I signal on normal epidermal keratinocytes. Each dot represents a tumor sample (n = 35 in each group, paired t test). FIG. 4E: Representative image of Foxcl and HLA-I stained BCC shown in its original form and following the analysis by HALO software to spatially resolve the distance between Foxcl and HLA-I expression in the cancer cells. Distance between Foxcl+ cells and HLA-I^mcdllim versus HLA-F^/low cells are measured in the nearest neighbor analysis. FIG. 4F: Quantification of the distance between Foxcl+ cells and HLA-I^mcdllim versus HLA-I-/low cells in nearest neighbor analysis. Each dot represents a tumor sample (n = 35 in each group, paired t-test). FIG. 4G: Schematic diagram showing the persistence of Foxcl -mediated HLA-I downregulation via epigenetic modification from hair follicle stem cells to BCC cancer cells over several mitoses, which govern BCC’s low immunogenicity. FIG. 4H: Representative images of CD4 and CD8 stained pediatric SCC and BCC. FIG. 41: Quantification of CD8+ T cells as percent DAPI+ cells in the parenchyma of pediatric SCC and BCC. Each dot represents a tumor sample (n = 4 in SCC and n = 5 in BCC group, Mann-Whitney U test). FIG. 4J: Representative images of Foxcl and HLA-I stained pediatric SCC and BCC. FIG. 4K: Quantification of HLA-I^hlgh cells as percent DAPI+ cells in the parenchyma of pediatric SCC and BCC. Each dot represents a tumor sample (n = 4 in SCC and n = 5 in BCC group, Mann-Whitney U test). Bar graphs show mean + SD, scale bars: 100 pm. FIGs. 4L and 4M: Representative flow cytometry histogram (FIG. 4L) and RFI (FIG. 4M) of HLA-I on the surface of UW- BCC1 cells treated with 0, 0.1, 1, or 10 pM entinostat (n = 4 in each group, one-way ANOVA with Dunnett’s multiple comparison test). FIGs. 4N and 40: Representative flow cytometry histogram (FIG. 4N) and RFI (FIG. 40) of P2M on the surface of UW-BCC1 cells transfected with Foxcl siRNA construct (siRNAFoxcl) versus control siRNA (siRNA-Ctrl) and treated with 1 pM entinostat or DMSO vehicle control (n = 6 in each group, one-way ANOVA with Dunnett’s multiple comparison test). Bar graphs show mean + SD.

FIG. 5: Entinostat induces antigen presentation in primary BCC. FIG. 5A: Representative images of H&E stained mouse BCC in the skin of Ptchl+/- mice after receiving DMBA plus UVB skin carcinogenesis protocol. FIG. 5B: Violin plot of Foxcl gene expression levels in epidermal keratinocytes in EHFP versus non-EHFP state. EHFP state was defined by the expression of Lhx2 and Lgr5 in keratinocytes. DESeq2 was used for differential gene expression (DGE) analysis and P value calculation. FIG. 5C: Representative images of P2M and Foxcl stained mouse BCC in Ptchl+/- skin treated with entinostat versus DMSO vehicle control. FIG. 5D: Representative images of CD8 stained Ptchl+/- skin treated with vehicle, entinostat, imiquimod, or entinostat plus imiquimod. Note that CD8+ cells are CD3+ T cells. FIG. 5E: Quantification of CD8+ T cells in Ptchl+/- skin treated with vehicle, entinostat, imiquimod, or entinostat plus imiquimod. Each dot represents a mouse (n = 15 in DMSO, n = 16 in entinostat, n = 9 in imiquimod, and n = 15 in entinostat plus imiquimod group, one-way ANOVA with Dunnett’s multiple comparison test). Bar graphs show mean + SD, scale bars: 100 pm.

FIG. 6: Foxcl expression in BCC is associated with quiescence, and the distribution of iatrogenic risk factors in pediatric SCC versus BCC patients. FIG. 6A: Representative images of Foxcl and Ki67 stained SCC and BCC. FIG. 6B: Quantification of Ki67+ cancer cells as percent Foxcl- versus Foxcl+ DAPI+ cells in the parenchyma of BCC. Each dot represents a tumor sample (n = 15 in each group, paired t-test ). FIG. 6C: Violin plots of FOXCI, HLA-A, HLA-B, HLA-C, and B2M gene expression levels in FOXC1+ versus FOXCI- BCC cancer cells. DESeq2 was used for DGE analysis and P value calculation. FIG. 6D: Venn diagrams of iatrogenic risk factors associated with pediatric SCC and BCC (n = 8 in SCC, n = 13 in BCC). Not displayed on the Venn diagram: Immunosuppression + Chemotherapy only, n = 0 in both SCC and BCC. Radiation + Voriconazole only, n = 0 in both SCC and BCC. Scale bar: 100 pm.

DETAILED DESCRIPTION

Previous clinical studies have demonstrated that organ transplant recipients (OTRs) treated with immunosuppressant therapy had 65-250 times higher risk of SCC development than in general population^{9, 10}. In contrast, BCC incidence is only 10 times higher in OTRs compared with the general population¹⁰. Furthermore, topical immunotherapy for actinic keratosis has demonstrated efficacy for SCC prevention but has no impact on BCC risk reduction¹¹.

These clinical observations indicate that a novel strategy is required to enable immunotherapy for BCC and other non-immunogenic tumors in the skin. Here, immunogenicity parameters between SCC and BCC have been compared, and provided herein is a novel therapeutic approach to induce BCC immunogenicity.

Provided herein is a previously unknown immunoevasion mechanism contributing to early cancer development: primary BCC has markedly lower immunogenicity compared with SCC even though BCC has higher TMB, an established determinant of tumor immunogenicity⁷. BCC shows an immune-excluded phenotype as T cells are confined to the stroma around the tumor foci while tumor parenchyma lacks immune cell infiltrate⁶². BCC cancer cells have low APM gene expression, which is the major cause of reduced cancer immunogenicity⁶²'⁶⁴. We find that Foxcl is a critical suppressor of IRF1 and HLA-I in BCC cells, reminiscent of its function in quiescent hair follicle stem cells³³. Foxcl downregulates IRF1 and HLA-I expression by epigenetic mechanisms, which can be reversed to enable BCC immunotherapy. This discovery explains the previously unrecognized role of the cell of origin in dictating cancer immunogenicity. Furthermore, it highlights the critical need to study early cancer immunology to develop effective cancer prevention and treatment modalities.

Foxcl is expressed by hair follicle stem cells as a regulator of cellular quiescence³³'³⁵. Foxcl expression in BCC cancer cells is associated with a lack of HLA-I and Ki67, characteristic of quiescent stem cells in hair follicles³³. In addition, we have discovered that Foxcl+ BCC cancer cells are located in the center of tumor foci. By contrast, cell proliferation and hedgehog pathway activity are preferentially elevated in the periphery of BCC tumor foci⁶⁵. These features are reminiscent of normal hair follicles in which Foxcl+ quiescent stem cells are segregated from proliferating progenitor cells^66,67. Although BCC’s cell of origin may vary^47,68, it is well-established that BCC can arise from hair follicle stem cells⁶⁹'⁷². These findings support the fact that BCC arises from Foxcl+ hair follicle stem cells. Furthermore, interfollicular keratinocytes in the EHFP state, which give rise to BCC⁴⁹, upregulate Foxcl. Like their cells of origin, BCC stem cells may maintain a quiescent state by sustaining Foxcl expression to retain sternness and prevent exhaustion⁷³'⁷⁵.

Based on the localization of Foxcl+ cells in the center of BCC tumor foci, we speculate that BCC quiescent cancer stem cells in the center of the tumor foci give rise to progenitor cells in the periphery. HLA-I^mcdllini BCC cancer cells in the periphery are located farthest from HLA-I'^/low Foxcl+ BCC tumor cells. Thus, we propose that HLA-I'^/low Foxcl+ BCC stem cells lose Foxcl expression as they proliferate and give rise to Foxcl- daughter cells expressing very low levels of HLA- I. Foxcl- BCC cells eventually recover some degree of HLA-I expression in the periphery after multiple cell divisions.

Foxcl downregulates APM gene expression by epigenetic modification, including histone deacetylation of the promoter region. To reverse this effect and transform BCC into an immunogenic cancer, we demonstrate that an HD AC inhibitor, entinostat, upregulates APM genes in BCC cells. Entinostat, also known as SNDX- 275 or MS-275, is a benzamind histone deacetylase inhibitor which inhibits class I HDAC1 and HDAC3, enzymes that regulates chromatin structure and gene transcription. Systemic modulation of DNA acetylation and methylation by HD AC and DNA methyltransferase inhibitors has been shown to promote cancer immunogenicity⁷⁶'⁷⁸. Although it upregulates APM, topical entinostat treatment alone does not affect BCC counts or T cell infiltration in vivo. As such, combining entinostat with topical and systemic immunotherapies is key to enabling BCC immunotherapy^79,80. To accomplish this, we have combined topical entinostat with an FDA-approved topical immunotherapy, imiquimod. Repeated topical application of imiquimod results in regression of superficial skin cancers through the induction of innate immunity and apoptosis of cancer cells ^81,82. In addition, imiquimod stimulates CD8+ T cells and increases their cytolytic enzymes to eliminate MHC-I-expressing cancer cells⁸³. The high efficacy of entinostat plus imiquimod combination in suppressing BCC development indicates that entinostat-induced APM expression in BCC cancer cells synergizes with immune cell activation by imiquimod to eliminate BCC in vivo.

Pharmaceutical Compositions

The methods described herein include the use of pharmaceutical compositions comprising an HD AC inhibitor for the treatment of non-immunogenic skin cancer, basal cell carcinoma (BCC), or Foxcl positive tumor. The pharmaceutical compositions can further include a TLR7/8 agonist, and/or a vitamin D analog, and/or 5-FU chemotherapy, and/or systemic immunotherapy.

In some embodiments, the methods include pharmaceutical compositions that contain an HD AC inhibitor such as panobinostat, abexinostat, entinostat, mocetinostat, vorinostat, pracinostat, ricolinostat, apicidin, fimepinostat, P300 histone acetylatransferase, tubastatin A, tucidinostat, romidepsin, belinostat, givinostat, or valproic acid. In some embodiments, the HD AC inhibitor comprises etinostat. In some embodiments, the HDAC inhibitor is administered systemically. In some embodiments, the HDAC inhibitor is administered topically.

In some embodiments, the methods include pharmaceutical compositions that contain a TLR7/8 agonist, such as imiquimod, resiquimod, 852-A, vesatolimod, AZD8848, motolimod, or selgantolimod. In some embodiments, the TLR7/8 agonist comprises imiquimod. In some embodiments, the TLR7/8 agonist is administered topically.

In some embodiments, the methods include pharmaceutical compositions that contain a vitamin D or an analog thereof, such as doxercalciferol, pari cal citol, calcitriol, tacal citol, alfacalcidol, calcijex, calcipotriol, falecalcitriol, dihydroxycholecalciferol, rayaldee, rocaltrol, calcifediol, cholecalciferol, decara, oleovitamin d3, or trionex. Vitamin D refers to a group of fat-soluble secosteroids. Secosteroids are very similar in structure to steroids except that two of the B-ring carbon atoms of the typical four steroid rings are not joined, whereas in steroids they are. Examples include active vitamin D (or calcitriol), and Vitamin D3, also known as cholecalciferol (a form of vitamin D generated in the skin of animals when light energy is absorbed by a precursor molecule 7-dehydrocholesterol). In some embodiments, the vitamin D analog comprises calcipotriol. Structurally modified derivatives of vitamin D may be referred to as vitamin D analogs. Vitamin D analogs may be modified to improve bioavailability, solubility, have improved stability and/or handling properties compared to an unmodified version. Prodrugs of vitamin D analogs are also contemplated. Any vitamin D analog capable of binding to vitamin D receptor and inducing thymic stromal lymphopoietin (TSLP) may be suitable for a composition as described herein. Vitamin D analogs are many, and will be recognized by the skilled person. Non-limiting examples of suitable vitamin D analogs include I, 24-(OH)2D3 (calcitrol), 26,27-Ffl-I,25-(OH)2D3 (ST- 630), I.alpha.-(OH)D2, Ia-(0H)D3, 1,24-(OH)2D3 (TV-02), 22-oxacalcitriol (OCT), calcipotriol (MC 903), I,25-(OH)2-16-ene-23-yne-D3 (Ro 23-7553), EB 1089, ED- 71, PRI-2191, PRI-2205, cholecalciferol, ergocalciferol, calciferol, Calcijex, calcitriol, doxercalciferol, Hectorol, paricalcitol, Rocaltrol, Daivonex, and Zemplar, and other analogs, e.g., as described in e.g., as described in US 6,753,013. The term “analog”, in the context of the present invention, is meant to include synthetic analogs as well as vitamin D metabolites.

In some embodiments, the vitamin D analog is calcipotriol (also known as calcipotriene, or calcitrene). Calcipotriene is a synthetic derivative of calcitriol, a form of vitamin D. It is used in the treatment of psoriasis. The induction of an epidermis- derived cytokine, thymic stromal lymphopoietin (TSLP), protects against skin and breast cancer development. This protection is mediated by T cells responding directly to TSLP. Calcipotriene, a low-calcemic vitamin D analog and inducer of TSLP, is an immunotherapy that has been used for breast cancer treatment. The methods can include the use of a single composition including all agents, or two or more compositions each comprising one or more of the agents. In some embodiments, these are the only active agents, i.e., no other active agents are included in the compositions or methods. Such compositions can be configured to control release of the multiple agents at different rates, either sequentially or concomitantly.

Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions; in some embodiments, however, no other active compounds are used. Some of the present compositions and methods do not include the use of cytotoxic agents, e.g., as described in US2017/0246299, and/or do not include the use of anti-inflammatory drugs, e.g., NSAIDS such as diclofenac, and/or do not include the use of steroidal compounds, e.g., hydrocortisone valerate.

Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include systemic, parenteral, e.g., intravenous, intradermal, intralesional/intratumoral, intrasubcutaneous; transdermal or topical; transmucosal; or rectal or vaginal administration.

Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

In some embodiments, which include a vitamin D analog, and the methods include topical application, a solvent is also included that promotes stability of the vitamin D/D analog, e.g., as described in US 6,753,013, non-limiting examples of which include: Arlamol E (polyoxyethylene(15) stearyl ether); Arlamol DoA (diisooctyl ester of adipic acid); Arlasolve 200 (Polyoxyethylene-20-isohexadecyl ether); Eutanol G (2-octyldodecanol); Finsolv (Isostearyl benzoate); Finsolv P (polyoxypropylene- 15 -stearyl ether benzoate); Isopropylesters of straight or branched C. sub.10 -C. sub.18 alkanoic or alkenoic acids such as isopropyl myristate, isopropyl palmitate, isopropyl isostearate, isopropyl linolate and isopropyl monooleate; Miglyol 840 (Propylene glycol diester of caprylic and caprinic acid); DPPG (propylene glycol dipelagonate); and Procetyl AWS.

Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Systemic administration of a therapeutic compound as described herein can be via injection or by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For topical or transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

In some embodiments, compositions comprising one or more agents for topical application can further comprise cosmetically-acceptable carriers or vehicles and any optional components. A number of such cosmetically acceptable carriers, vehicles and optional components are known in the art and include carriers and vehicles suitable for application to skin (e.g., ointments, sunscreens, creams, milks, lotions, masks, serums, etc.), see, e.g., U.S. Patent Nos. 6,645,512 and 6,641,824. In particular, optional components that may be desirable include, but are not limited to absorbents, anti-acne actives, anti-caking agents, anti-cellulite agents, anti-foaming agents, anti-fungal actives, anti-inflammatory actives, anti-microbial actives, antioxidants, antiperspirant/deodorant actives, anti-skin atrophy actives, anti-viral agents, anti-wrinkle actives, artificial tanning agents and accelerators, astringents, barrier repair agents, binders, buffering agents, bulking agents, chelating agents, colorants, dyes, enzymes, essential oils, film formers, flavors, fragrances, humectants, hydrocolloids, light diffusers, nail enamels, opacifying agents, optical brighteners, optical modifiers, particulates, perfumes, pH adjusters, sequestering agents, skin conditioners/moisturizers, skin feel modifiers, skin protectants, skin sensates, skin treating agents, skin exfoliating agents, skin lightening agents, skin soothing and/or healing agents, skin thickeners, sunscreen actives, topical anesthetics, vitamin compounds, and combinations thereof. In some embodiments, the compositions include one or more of benzyl alcohol, cetyl alcohol, glycerin, isostearic acid, methylparaben, polysorbate 60, propylparaben, purified water, sorbitan monostearate, stearyl alcohol, white petrolatum, and/or xanthan gum.

Topical compositions are suitable for safe and effective use within a range of concentrations. Accordingly, the amount of topical active agent in compositions described herein is between 5 ug/mL and 2000 ug/mL, for example, 25 ug/mL, 100 ug/mL, 500 ug/mL 1000 ug/mL, 1500 ug/mL or 2000 ug/mL. In specific embodiments, calcipotriene (0.005% ug/mL) and about 5% ug/mL to about 3.75% ug/mL imiquimod are used in combination. The dosage schedule for this use, i.e., the dosing regimen, will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient’s physical status, age and the like. In some embodiments, dosing should continue for at least 1 month.

The topical compositions should also be suitable for safe and effective use within a pH range. Accordingly, in some embodiments, the pH of topical compositions described herein is between 6.0 and 7.5.

In some embodiments, compositions as described herein are suitable for topical administration. Methods as described herein are ideally suited for increasing an amount of at least one agent in the epidermis, or epidermis and dermis, within a region of a subject’s skin. Topical compositions described herein can be administered in dosages suitable to enter the blood stream. Accordingly, agents can be administered topically, as adjuvants, prior to, or concomitantly with, systemic administration directly into the blood stream.

The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. A composition can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture. Compositions may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, ointments, pastes, emulsions, sprays, creams, lotions, slurries, suspensions, foams, controlled release compositions, gels, patches, implants, water-oil bilayer compositions, water-oil-powder trilayer compositions, serums, powders, mousses, hydrogels, single-use applicators, and the like, suitable for topical administration to the patient.

The carrier can be in a wide variety of forms. Non-limiting examples include simple solutions (e.g., aqueous, organic solvent, or oil based), emulsions, and solid forms (e.g., gels, sticks, flowable solids, or amorphous materials). In certain embodiments, the dermatologically acceptable carrier is in the form of an emulsion. Emulsion may be generally classified as having a continuous aqueous phase (e.g., oil- in-water and water-in-oil-in-water) or a continuous oil phase (e.g., water-in-oil and oil-in-water-in-oil). The oil phase of the present invention may comprise silicone oils, non-silicone oils such as hydrocarbon oils, esters, ethers, and the like, and mixtures thereof.

Emulsions may further comprise an emulsifier. The composition may comprise any suitable percentage of emulsifier to sufficiently emulsify the carrier. Suitable weight ranges include from about 0.1 wt % to about 10 wt % or about 0.2 wt % to about 5 wt % of an emulsifier, based on the weight of the composition. Emulsifiers may be nonionic, anionic, or cationic. Suitable emulsifiers are disclosed in, for example, U.S. Patent Nos. 3,755,560 and 4,421,769, and McCutcheon's Detergents and Emulsifiers, North American Edition, pages 317-324 (1986), which are incorporated herein by reference in their entirety. Suitable emulsions may have a wide range of viscosities, depending on the desired product form. Non-limiting examples of emulsifiers include glyceryl stearate, polysorbate 60, and the PEG- 6/PEG-32/glycerol stearate mixture sold under the name of Trefose® by Gattefosse. An emulsion may contain a fatty phase that may range from between about 5 wt % to about 80 wt % (e.g., between about 5 wt % to about 50 wt %) of the composition. Any of the emulsions described herein may contain one or more agents selected from the group of oils, waxes, emulsifiers, and coemulsifiers. Examples of oils, waxes, emulsifiers, and coemulsifiers used in compositions are well-known in the art. An emulsifier and a coemulsifier may be present in the composition in a proportion ranging from 0.3 wt % to about 30 wt % (e.g., between about 0.5 wt % to about 20 wt %) of the composition. An emulsion may contain lipid vesicles.

Other agents that can be administered together with, or following administration, include but are not limited to, antimicrobial agents, antiseptic agents and analgesic agents. Such agents can be administered sequentially (e.g., after prior administration of topical compositions) or concomitantly, for example, in a dual release dosage composition.

Methods of Treatment

Provided herein are methods for treating a non -immunogenic skin cancer (e.g., Extramammary Paget’s disease, chest wall metastasis of breast cancer, malignant melanoma, and adnexal carcinoma), basal cell carcinoma (BCC), or Foxcl positive tumor in a subject in need thereof, the method comprising administering a therapeutically effective amount of an HD AC inhibitor along with one or more of a TLR7/8 agonist, a vitamin D analog, a chemotherapy agent, and a systemic immunotherapy.

Methods for treating a mammal having a non-immunogenic skin cancer, BCC, or Foxcl positive tumor can include identifying the mammal as having a non- immunogenic skin cancer, BCC, or Foxcl positive tumor. Examples of methods for identifying the mammal as having non-immunogenic skin cancer, BCC, or Foxcl positive tumor include, without limitation, physical examination, laboratory tests (e.g., blood or urine), biopsy, endoscopy, genetic tests, or other methods as known in the art.

The methods can also include administration of one or more other treatments known in the art for non-immunogenic skin cancers, BCCs, or Foxcl positive tumors. For example, a combination treatment with the methods described herein can include anti-inflammatory drugs, corticosteroids, pain-killing medication, or immunosuppressant drugs. Examples of anti-inflammatory drugs include, without limitation, diclofenac, naproxen, flurbiprofen, celecoxib, fenoprofen, tolmetin, diflunisal, ibuprofen, etodolac, nabumetone, ketoprofen, mefenamic acid, ketorolac, magnesium salicylate, indomethacin, aspirin, piroicam, sulindac, meloxicam, or oxaprozin. Examples of corticosteroids include, without limitation, methylprednisolone, betamethasone, budesonide, decadron, pulmicort, entocort EC, dexamethasone, hydrocortisone, alclometasone, dexamethasone intensol, beclomethasone, prednisolone, triamcinolone, cortisone acetate, fluticasone propionate, or clobetasol. Examples of pain-killing medication include, without limitation, morphine, diclofenac, aspirin, meperidine, acetaminophen, piroxicam, oxycodone, hydrocodone, tramadol, ketorolac, indomethacin, naproxen, ibuprofen, hydromorphone, or methadone. Examples of immunosuppressant drugs include, without limitation, mycophenolate mofetil, cyclosporine, everolimus, abatacept, adalimumab, anakinra, certolizumab, entercept, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, vedolizumab, golimumab, ATGAM, ASTAGRAF XL, mycophenolic acid, azathioprine, sirolimus, leflunomide, ciclosporin, methotrexate, muromonab, basiliximab, prednisone, tacrolimus, omalizumab, daclizumab, alemtuzumab, or fmgolimod.

In cases where the HD AC inhibitor (e.g., entinostat) and additional agents are provided separately, the administration of HD AC inhibitor (e.g., entinostat) can be in any order relative to the administration of one or more agents. For example, HDAC inhibitor (e.g., entinostat) can be administered to a mammal prior to, concurrent with, or following administration of one or more additional agents.

The HDAC inhibitor (e.g., entinostat) and/or additional agents are administered in a therapeutically effective amount. A “therapeutically effective amount” is an amount sufficient to effect beneficial or desired results. For example, an effective amount is one that achieves a desired therapeutic effect, e.g., an amount necessary to treat a disease, or to reduce risk of development of disease or disease symptoms (also referred to as a prophylactically effective amount). An effective amount can be administered in one or more administrations, applications, or dosages. A therapeutically effective amount of a therapeutic compound (i.e., an effective dosage) depends on the therapeutic compounds selected. For example, a therapeutically effective amount HDAC inhibitor provided herein can be effective to reduce the number of or severity of symptoms in a mammal (e.g., human). The HDAC inhibitor can be administered one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments. Various factors can influence the actual amount used for a particular application. For example, the frequency of administration, duration of treatment, combination of other agents, site of administration, stage of disease (if present), and the anatomical configuration of the treated area may require an increase or decrease in the actual amount administered.

The frequency of administration of HDAC inhibitor provided herein can be any frequency. For example, the frequency of administration can be from about four times a day to about once a month, or more specifically, from about twice a day to about once a week. In addition, the frequency of administration can remain constant or can be variable during the duration of treatment. As with the amount administered, various factors can influence the actual frequency of administration used for a particular application. For example, the amount (dose), duration of treatment, combination of agents, site of administration, stage of disease (if present), and the anatomical configuration of the treated area may require an increase or decrease in administration frequency. For example, the methods can include administering a first dose, followed by one or more additional doses at a later time (e.g., a “booster” dose), e.g., 1, 2, 3, 4, 5, 6, or 7 days from a previously administered dose. For example, administration can include administering two, three, four, or five doses, administered 1, 2, 3, 4, 5, 6, or 7 days apart. The length of time between multiple doses can be the same (e.g., 2 days between each dose, 3 days between each dose, 4 days between each dose, etc.), or the length of time between multiple doses can be different between each dose (e.g., 4 days between the first and second dose, 3 days between the second and third dose, and 2 days between the third and fourth dose).

In some embodiments, the methods include administration of a treatment described herein daily, e.g., once or twice daily, for at least 2, 3, 4, 5, 6, 7, 8 9, 10, 11, 12, 13 or 14 days, e.g., for 4 days, before surgical excision of the remaining lesion, e.g., at 7, 10, 12, 14, 21, or 28, e.g., 7-28, 14-21, days post treatment. In some embodiments, the methods include repeating the treatment regimen at least two, three, four, five, six or more times before optional surgical intervention. In some embodiments, a treatment regimen includes a treatment interval including administration of a treatment described herein daily, e.g., once or twice daily, for at least 2, 3, 4, 5, 6, 7, 8 9, 10, 11, 12, 13 or 14 days, e.g., for 4 days, and then a resting interval, e.g., of at least 7, 10, 12, 14, 21, or 28, e.g., 7-28, 14-21, days, between treatment intervals.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. The following methods were used to generate the examples.

Human cancer tissue study

De-identified adult BCC and SCC tissues, anatomical location, age, and gender of the patients were included in the study. Pediatric SCC and BCC tissue and information were collected as part of the multicenter, retrospective, case-control study of patients <20 years of age diagnosed with nonmelanoma skin cancer from Boston Children's Hospital60. Pediatric subjects’ consents were obtained, and tissues were analyzed in accordance with the Institutional Review Board at Boston Children's Hospital. The study of deidentified adult human BCC and SCC samples was reviewed and approved by the Institutional Review Board at Massachusetts General Hospital.

Cell lines and culture

Human BCC cell line, UW-BCC1, was provided by Dr. Vladimir Spiegelman (Pennsylvania State University, University Park, Pennsylvania)²³. Human cutaneous SCC cell lines, SCC12 and SCC13, were provided by Dr. James G Rheinwald (Brigham and Women’s Hospital, Boston, Massachusetts)^24,25. KERTr, a normal human keratinocyte cell line, was purchased from ATCC (CRL-2309). HaCaT, a normal human keratinocyte cell line, was provided by Dr. Anna Mandinova (Massachusetts General Hospital, Boston, MA). UW-BCC1, SCC12, SCC13, and HaCaT were cultured in DMEM medium (Thermo Fisher Scientific, Waltham, MA 11-965-118), including 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% glutamine, at 37°C in 5% CO2 supplemented air. KERTr was cultured in Keratinocyte-SFM media (Gibco, 17005-042), including bovine pituitary extract, recombinant epidermal growth factor, and 1% penicillin/streptomycin, at 37°C in 5% CO2 supplemented air.

Histology

Tissue samples were fixed with 4% paraformaldehyde (PF A) and embedded in paraffin. 5 pm sections were cut and deparaffinized. After being permeabilized with 0.2% Triton-X (Thermo Fisher Scientific, BP151) in phosphate-buffered saline (PBS) for 5 min, antigen retrieval was performed using a pressure cooker in citrate-based antigen unmasking solution (Vector Laboratories, Burlingame, CA, H-3300-250) or tris-based antigen unmasking solution (Vector Laboratories, H-3301-250) for 20 min. Slides were rinsed once in DI water and PBS, including 0.1% Tween 20 (Sigma- Aldrich, P1379) (PBS-T). Slides were blocked with 5% normal goat serum (Sigma- Aldrich, G9023) in PBS and incubated in a humidified chamber at room temperature for 1 h. Slides were incubated overnight at 4°C with primary antibodies diluted in the blocking buffer. When stained with Foxcl antibody or two primary antibodies from the same host animals, signal amplification and heat-induced stripping for antibodies were performed following the standard protocol using Opal 4-Color IHC Kit (PerkinElmer, Waltham, MA, NEL820001KT). Following overnight primary antibody application, slides were rinsed once and washed three times for 2 min each in PBS-T. Slides were incubated in secondary antibodies and 4’,6-diamidino-2- phenylindole (DAPI, Invitrogen, D3571, 1 :4000) diluted in the blocking buffer for 60 min at room temperature. Slides were washed in PBS-T and mounted with Fluoroshield (Sigma-Aldrich, F6182). The stained tissues were imaged with a ZEISS Axio Scan.Zl Slide Scanner (Zeiss, Oberkochen, Germany). Automated counting was performed using HALO 3.0 software (Indica Labs, Albuquerque, USA). Cell counts were reported as % positive cells per total DAPI positive cells in tumor parenchyma across five randomly selected high-power fields (hpf, 200x magnification) per sample in each group. The cut-offs for HLA-Ihigh and HLA-Imedium were determined to be 80% and 50% of the average HLA-I signal of the adjacent normal epidermis, respectively. For hematoxylin and eosin staining, slides were stained according to standard procedures and mounted with Cytoseal XYL (Thermo Fisher Scientific, 8312-4).

RNA-Seq analysis

Human skin cancer cell lines and keratinocyte cell lines were lysed with RNA Lysis Buffer, and RNA was isolated following the protocol of Quick-RNA Miniprep Kit (Zymo Research, R1055) and quantified using Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA). Libraries were prepared by Novogene (Sacramento, CA) using the NEBNext Ultra RNA Library Prep kit for Illumina (New England Biolabs, Ipswich, MA, E7770). Sequencing was performed by Novogene using the Illumina NovaSeq6000 System. Reads were aligned to the human reference genome (hg38) using STAR. Differential expression analysis was performed by Novogene using the DESeq2R package. Original data are available in the NCBI Gene Expression Omnibus (GEO) with accession number GSE236291 (RNA-Seq). scRNA-Seq analysis

The human BCC scRNA-Seq dataset was deposited under the accession code: GSE141526 and previously described^87,88. The human SCC scRNA-Seq dataset was deposited under the accession code: GSE193304 and previously described⁸⁹. The Mouse scRNA-Seq dataset was deposited under the accession code: GSE228047 and previously described⁴⁹. Briefly, human cancer specimens were surgically excised, and tumor digestion was performed using either diapase II (Sigma-Aldrich), collagenase IV (Sigma-Aldrich), and trypsin-EDTA or tumor dissociation kit for human (130-095- 929, MACS Miltenyi Biotec). After excluding debris and dead cells, cells were captured using Chromium (10X Genomics). Mouse BCC tumor tissue was collected and digested at 6 weeks after oncogene expression, and SmoM2 -mutated cells were isolated based on the expression of the YFP fused to the oncogene with the exclusion of hair follicle bulge cells. For mouse BCC and human SCC, the single-cell capturing and downstream library constructions were performed using the Chromium Single Cell 3' v3 library preparation kit (lOx Genomics) according to the manufacturer’s protocol, and the libraries were sequenced on NovaSeq6000 (Illumina). For human BCC, the single-cell capturing and downstream library constructions were performed using the Chromium Single Cell 3' v2 library preparation kit (lOx Genomics) according to the manufacturer’s protocol, and the libraries were sequenced on the Illumina Hi Seq4000 platform (Illumina).

The FASTQ files were aligned using human reference (GRCh38) with the Cell Ranger count function (6.0). Quality control and downstream analysis were performed using the Seurat R package (v.4.3.O)⁹⁰. For each sample, all cells passed the following criteria: showed expression of more than 20 and fewer than 3,000 unique genes and had less than 10% unique molecular identifier (UMI) counts belonging to mitochondrial sequences. Read counts were normalized by the NormalizeData function of Seurat with the default parameter. Principal component analysis (PCA) for each sample was calculated using the scaled expression data of the most variable genes (identified as outliers on a mean/variability plot, implemented in the FindVariableGenes). UMAP calculation and graph-based clustering were done for each sample using the appropriate functions from Seurat (default parameters) with the respective PCA results as input. The batch effect among samples was removed using the "SelectlntegrationFeatures" and "FindlntegrationAnchors" functions. The gene sets for identifying cells with EHFP signature have been defined previously^49,50. To visualize the AUC score calculated by AUCell, the AUC score matrix was embedded into each dataset as an additional assay with the CreateAssayObject. Flow cytometry

Cells were washed once with PBS, including 5% newborn calf serum (Thermo Fisher Scientific, 26010074) and 0.01% sodium azide (Sigma-Aldrich, S2002-100G), and stained with antibodies on ice for 30 min. Following the surface marker staining, cells were fixed and permeabilized for intracellular staining using Fixation Buffer (BioLegend, 420801) and Intracellular Staining Perm Wash Buffer (BioLgend, 421002) when intracellular markers were evaluated. Permeabilized cells were stained with antibodies overnight at 4 °C. Cells were washed and examined by BD LSRFortessa X-20 flow cytometer (BD Bioscience, Billerica, MA). Data were analyzed using FlowJo software (BD Bioscience).

Drug treatment in vitro

Cells were treated with entinostat (Selleck Chemicals, Houston, TX, SI 053) at the concentration of 1 pM for three days in serum-reduced DMEM medium (Thermo Fisher Scientific), including 1% FBS to reduce analytical interference and provide more reproducible experimental conditions unless indicated otherwise⁹¹. Cells were treated with 100 nM vismodegib (ApexBio, A3021), 10 nM romidepsin (Advanced ChemBlocks Inc, Hayward, CA, Cat#M15388), 1 pM remetinostat (MedChemExpress, Monmouth Junction, NJ, Cat#HY-100365), 0.1 pM givinostat (Cayman Chemical, Ann Arbor, MI, Cat# 11045), or 1 pM vorinostat (Cayman Chemical, Cat#10009929) for three days in serum-reduced DMEM medium.

Quantitative PCR

RNA was isolated following the protocol of Quick-RNA Miniprep Kit (Zymo Research, R1055) and quantified using Nano-Drop ND-1100 (NanoDrop Technologies, Wilmington, DE). cDNA samples were synthesized from 1 pg of total RNA using Invitrogen SuperScripts III Reverse Transcriptase (Thermo Fisher Scientific, 18080085). Expression levels of all cDNA samples were determined with QuantStudio 5 (Thermo Fisher Scientific) using iTaq Universal SYBR green supermix (Bio- Rad Laboratories, Hercules, CA, 1725121). Quantitative real-time PCR for SYBR green analyses were performed in a final reaction volume of 10 pl consisting of 2 pl of cDNA of the respective sample and 8 pl of SYBR green master mix mixed with the corresponding primers (2 pM) for each gene. All assays were conducted in triplicate and normalized to GAPDH expression.

ChlP-qPCR assay

UW-BCC1 cells that had either been transfected with siRNA construct to knock down Foxcl or control were fixed in 1% formaldehyde (Millipore Sigma, F8775) for 10 min and were washed in cold PBS. Cells were lysed with buffer containing 2.5% glycerol, 50 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40, and 0.25% Triton X-100. After centrifugation at 400 g, cell lysates were resuspended in buffer A containing 1 mM Tris-HCl (pH 7.9), 20 mM NaCl, and 0.5 mM EDTA and incubated at room temperature for 10 min. Following the second centrifugation at 400 g, cells were sonicated in sonication buffer containing 10 mM HEPES, 1 mM EDTA, and 0.5% SDS for 30 min to achieve chromatin fragmentation. Following centrifugation at 16,200 g, proteins were immunoprecipitated with anti- Foxcl or anti-H3K27ac antibody at 4°C overnight. The next day, the samples were incubated with 30 pl 50% slurry of ChlP-Grade Protein G agarose beads (Cell Signaling Technology, 9007S) for 2 h. Samples were precipitated and washed three times with wash buffer containing 20 mM Tris-HCl (pH 7.9), 10 mM NaCl, 2 mM EDTA, 0.1% SDS, and Triton X-100. After the last wash step, samples were eluted with 100 pl of TE buffer containing 100 mM Tris-HCl (pH 8), 1 mM EDTA, and 1% SDS three times. De-crosslinking was performed by overnight incubation with 15 pl of 3 M NaCl at 65°C. Precipitated DNA was purified by ChIP DNA Clean & Concentrator (ZYMO research, Irvine, CA, D5205) and subjected to quantitative PCR.

Western blot

Cell lysates were prepared in RIPA buffer (Thermo Fisher Scientific, 89900) consisting of a IX protease inhibitor cocktail, EDTA-free (Thermo Fisher Scientific, A32955). Cell lysates were sonicated for 20 s and subsequently centrifugated at 13,300 g. After checking the protein concentration of each sample using BCA Protein Assay kits (Thermo Fisher Scientific, P123227), the same amount of protein was loaded into Mini -PROTEIN TGXTM Gels (BIO-RAD, Hercules, 456- 1083 or 456- 1086) with IX Tris/Glycine/SDS buffer (BIO-RAD, catalog no. 1610732). A few minutes later (according to protein size), they were transferred to Immobilon-P membrane (Millipore Sigma, IPVH00010) with Transfer buffer (Boston Bioproducts, Ashland, MA, BP- 190). Then, they were incubated with 3% bovine serum albumin (Thermo Fisher Scientific, BP 1600) or 5% Skim milk (BD biosciences, San Jose, CA, 232100) in IX Tris-Buffered Saline (Boston Bioproducts, catalog no. BM301X) containing 0.1% Tween (TBS-T) for 30 min. After the membrane was washed with TBS-T, they were subjected to immunoblot with proper antibodies overnight at 4°C. The following day, they were incubated with appropriate secondary antibodies and were developed with a Pierce ECL Western blotting substrate kit (Thermo Fisher Scientific, 32106). Western blot bands were quantified with cSeries Capture software (Azure biosystem, Dublin, CA, Azure 600). Each band quantity was calculated by measuring band intensity minus the background. Total proteins were normalized based on GAPDH levels. Pictures of full Western blot gels are provided as a source data file. siRNA transfection

LTW-BCC1 cells were seeded at a density of 5 * 10⁴ cells into 12-well plates. One day after seeding, UW-BCC1 cells were transfected with siRNA-Foxcl knockdown construct. Negative Control siRNA (Qiagen, 1022076) was used for negative control. The final siRNA concentration was 10 nM. Transfections were performed using Lipofectaine RNAiMax according to the manufacturer’s instructions (ThermoFisher Scientific, 13778500). 48 h after transfection, cells were collected for flow cytometric analysis and quantitative PCR analysis. When combined with entinostat treatment, transfection was started 24 h after entinostat treatment. 24 h after transfection, cells were collected for flow cytometric analysis.

Lentivirus transduction

Lentivirus ORF particles, IRF1 (mGFP-tagged) (OriGene Technologies, RC203500L4V) was used. The lentivirus particle was incubated with LTW-BCC1 cells with 6 pg/ml polybrene (MilliporeSigma, TR-1003-G) for 4 hours on a 24-well plate. The cells were added with 250 pl of complete media and cultured in a cell incubator. Two days after transduction, 3 pg/ml puromycin was added for selection. After two passages, the cells were collected and confirmed to be GFPpositive by flow cytometry.

Animal studies

All mice were housed under pathogen-free conditions in an animal facility at Massachusetts General Hospital in accordance with animal care regulations. Ptchl+/- mice were purchased from Jackson Laboratory (J AX stock #003081)⁹². Mutant mice were genotyped. Age- and gender-matched mice were used in all experiments. At 8- 10 weeks of age, one day after the back skin hair was shaved, mice received 100 pg 7,12- dimethylbenz(a)anthracene (DMBA, Sigma-Aldrich, Cat#D3254-lG) in 200 pl acetone on the back skin (Week 0). After one week, mice were exposed to 250 mJ/cm2 ultraviolet B (UVB) 3 times per week for up to 20 weeks using a UVP XX- 15MR Bench Lamp, 302 nm (Analytik Jena, Jena, Germany, 95004208). The lamp was calibrated using a digital light meter (International Light Technologies, ILT1400A). Starting at 15 weeks post-DMBA, mice received topical entinostat (5 pM) in 200 pl of acetone, imiquimod (50 pg), entinostat plus imiquimod or control treatment twice a week on their back skin until the conclusion of the study at 20 weeks post-DMBA.

Statistical Analysis

All bar graphs show mean + SD. The two-tailed Mann-Whitney U test was used as the significance test for unpaired two-group comparisons. A two-tailed paired Ltest was used for two paired group comparisons. One-way ANOVA with Dunnett’s multiple comparison test was used for comparison between more than two groups. Clinical study analyses were performed using 2-sample Student’s Ltest and Pearson’s %2 tests on proportions in contingency tables. GraphPad Prism 10 was used for statistical analysis. P value < 0.05 was considered significant.

Example 1: Primary human BCC has low immunogenicity associated with low APM expression in cancer cells

To determine BCC’s immunogenicity, we examined the immune cell infiltrates in 35 primary nodular BCCs compared to 35 primary cutaneous SCCs, which also originated from sun-damaged keratinocytes in the skin. Although T cells were detectable in the stroma surrounding the BCC, significantly fewer CD4+ T and CD8+ T cells infiltrated into BCC tumor foci compared with SCC (FIGs. 1 A-1C). A survey of other immune cell types revealed that CD1 lc+ dendritic cells, CD207+ HLA-II+ Langerhans cells, and CD56+ natural killer (NK) cells were significantly reduced in BCC compared with SCC (FIGs. 1K-1P). Consistent with these findings, single-cell RNA sequencing (scRNA-Seq) analysis showed much smaller immune cell populations, including T cells, in BCC compared with SCC (FIG. ID). Furthermore, the percentage of CD8+ T cells was significantly lower in the T cell cluster of BCC compared with SCC (FIG. IQ). These findings indicate that primary BCC has an “immune excluded” phenotype, which leads to low immunogenicity of BCC compared with “immune infiltrated” SCC²¹.

APM downregulation is a major mechanism by which cancers evade antitumor immunity^19,22. Interestingly, primary BCC cells showed markedly lower HLA-I and P2M expression than SCC and the adjacent epidermal keratinocytes (FIGs. IE, IF, 1R, and IS). To examine whether low HLA-I expression in BCC tumor cells represents an acquired immune evasion mechanism (i.e., HLA-I downregulation) under selection pressure from CD8+ T cells¹⁹ or it is intrinsic to the nature of BCC development, we examined early BCC lesions in the skin. Importantly, early BCC with rare T cells in the stroma showed low HLA-I expression (FIG. 1G), indicating that low APM levels in BCC originate from an intrinsic mechanism integral to its biology.

To investigate the mechanism of HLA-I suppression in BCC, we examined the gene expression profile of a human BCC cell line (UW-BCC1)²³ in comparison with two human SCC (SCC12 and SCC13) and two human normal keratinocyte (KERTr and HaCaT) cell lines using RNA sequencing (RNA-Seq)²³'²⁷. UW-BCC1 cells expressed lower levels of APM genes broadly compared with SCC cell lines (FIG. 1H). In addition, HI.A-A, B2M, TAPI, TAP2, TAPBP, TAPBPL, ERAP1, and ERAP2 expression were lower in UW-BCC1 cells compared with normal keratinocyte cell lines (FIG. 1H). Consistently, UW-BCC1 showed markedly lower expression of HLA-I and P2M proteins on the cell surface compared with SCC and normal keratinocyte cell lines (FIGs. II, 1J, IT, and 1U). Thus, BCC’s low immunogenicity can be explained by the lack of antigen presentation, an intrinsic characteristic of BCC cancer cells. Example 2: Forkhead Box Cl (Foxcl) downregulates antigen presentation n BCC cancer cells

To determine the mechanism of HLA-I suppression in BCC, we examined the regulation of HLA-I gene expression. First, we investigated whether sonic hedgehog pathway activation, the main driver of malignant transformation in BCC^28,29, suppressed HLA-I expression in BCC cancer cells³⁰. Blocking the sonic hedgehog pathway with vismodegib did not alter HLA-I expression in UWBCC1 cells (FIG. IV). There are three major transcription factor binding sites controlling HLA-I heavy chain genes: an enhancer A region recognized by NF-kB, an interferon-stimulated response element (ISRE) bound by interferon regulatory factor (IRF) 1, and an SXY- module recognized by NOD-like receptor family CARD domain containing 5 (NLRC5)^22,31’³². We found that IRF1 was significantly downregulated while NLRC5 and phosphorylated NF-kB p65 were not altered in UW-BCC1 cells compared with SCC and normal keratinocyte cell lines (FIG. 2A-C, 2Q-2S). Foxcl is a transcription factor that plays an essential role in maintaining quiescent hair follicle stem cells, and its loss has been associated with increased antigen presentation in hair follicle stem cells³³'³⁵. Considering that hair follicle stem cells can be the origin of BCC^36,37, we examined Foxcl levels in BCC cells. Foxcl was highly expressed in UW-BCC1 compared to SCC and normal keratinocyte cell lines (FIG. 2A, 2D, and 2Q). Next, we investigated whether IRF 1 and Foxcl regulated HLA-I expression in BCC cells. IRF1 overexpression and Foxcl knockdown in UW-BCC1 led to upregulation of HLA-I and P2M (FIGs. 2E-2H, and 2T-2AA). IRF1 overexpression in UW-BCC1 did not inhibit Foxcl expression (FIGs. 21 and 2J). By contrast, Foxcl knockdown significantly upregulated IRF1 levels in UWBCC1 cells (FIGs. 2K and 2L). Foxcl knockdown upregulated APM genes at the transcriptional level (FIG. 2BB-2FF).

To determine whether Foxcl directly regulates HLA-I and IRF1 gene expression, we examined Foxcl binding to the promoter region of APM genes. The core consensus forkhead response element (FHRE) sequence is “RYMAAYA” (R = A/G; Y = C/T; M = A/C)³⁸. Foxcl chromatin immunoprecipitation-sequencing (ChlP- Seq) dataset (ERP006190) analysis has shown that 32.3% of total Foxcl binding sites contain RYAAACA (R = A/G; Y = C/T) motif³⁹. Foxc2, which shares 98% sequence homology in the DNA-binding domain with Foxcl, recognizes consensus sequence GTAAACA with the highest affinity³⁹. Another Foxc DNA-binding site is ATAAACA⁴⁰. Predicted Foxcl DNA-binding sites, ATAAACA/GTAAACA, were present at -6 kilobases (kb), -0.5 kb, -0.4 kb upstream of IRF1, HLA-A, and B2M transcription start sites, respectively. We generated primer sets flanking the Focxl DNA-binding sites for ChlP-quantitative PCR (ChlPqPCR) assay. Immunoprecipitation of endogenous Foxcl protein revealed its binding to the promoter regions of IRF1, HLA-A, and B2M genes in UW-BCC1 cells (FIG. 2M-2O). These results suggest that Foxcl suppresses antigen presentation by inhibiting IRF1 expression and directly blocking HLA-I and B2M expression by binding to their promoter region (Figure 2P). Thus, Foxcl is a master regulator of APM gene expression in BCC cancer cells.

Example 3: Entinostat reverses APM down regulation by Foxcl in BCC cancer cells

Foxcl transcriptional repression is associated with the recruitment of histone deacetylase (HD AC) 1 and 2 to the promoter region of target genes⁴¹'⁴⁴. Foxcl knockdown increased histone H3 lysine 27 acetylation (H3K27ac) at Foxcl -binding sites in the promoter region of HLA-A, B2M, and IRF1 (FIGs. 3 A-3B, and 3N). Based on this finding, we hypothesized that Foxcl-induced histone deacetylation can be reversed by HDAC inhibitors (FIG. 3C). Among several HDAC inhibitors, Entinostat, a class I HDAC inhibitor⁴⁵, showed the strongest activity in inducing HLA-I expression on the surface of UW-BCC1 cells (FIGs. 3D-3E, and 3O-3P). Entinostat also upregulated P2M, IRF1, and HLA-II levels on the surface of UW-BCC1 cells (FIG. 3Q-1). Entinostat treatment induced mRNA expression of HLA-A, HLA-B, HLA-C, and B2M (FIGs. 3W-3Z). By contrast, entinostat did not induce HLA-I, P2M, and IRFl expression in SCC12 cells (FIGs. 3AA-3FF). Entinostat impact on HLA-I induction in UW-BCC1 cells was dose-dependent, and this effect reached a plateau at the concentration of 1 pM (FIGs. 4L-4M). Importantly, Foxcl knockdown in UW- BCC1 cells treated with entinostat showed no further increase in HLA-I and P2M expression (FIGs. 3F-G and 4N-4O). These results demonstrate that entinostat treatment upregulates APM gene expression in BCC cancer cells, at least in part by reversing Foxcl transcriptional repression. Example 4: Entinostat upregulates antigen presentation in primary BCC and potentates topical immunotherapy for BCC

To determine the effect of entinostat treatment on BCC immunogenicity in vivo, we studied an established BCC model, Ptchl+/- mice, which spontaneously develop BCC resembling human BCC in response to ultraviolet B (UVB) radiation 28,29,46 ^_c/₂/+/_ _mice received 7,12-dimethylbenz(a)anthracene (DMBA) once, followed by UVB three times a week for 20 weeks to induce BCC (FIGs. 3H and 5A). In addition to hair follicle stem cells, which express Foxcl33-35, BCC can originate from interfollicular epidermis^47,48. BCC’s rise from the interfollicular epidermis has recently been found to originate from keratinocytes reprogrammed into an embryonic hair follicle progenitor (EHFP) state^49,50. Interestingly, the scRNA-Seq analysis revealed that epidermal keratinocytes in EHFP state upregulated Foxcl (FIG. 5B). Reminiscent of their cellular origin, cytokeratin 17 (CK17)-expressing primary BCCs in Ptchl +/- skin showed high nuclear Foxcl expression (FIG. 31)⁵¹'⁵³. Notably, topical entinostat treatment twice a week from week 15 to 20 post-DMBA significantly upregulated P2M expression in Ptchl+/- BCCs, including in Foxcl^hlgh cancer cells (FIGs. 3 J, 3K, and 5C). To determine whether entinostat can enable BCC immunotherapy, we combined entinostat with imiquimod, a topical Toll-like receptor 7 agonist used for BCC treatment⁵⁴. Ptchl+/- mice received topical entinostat, imiquimod, versus entinostat plus imiquimod combination on the back skin twice weekly from week 15 to 20 post-DMBA (FIG. 3H). Although entinostat and imiquimod monotherapy showed a limited effect on BCC development, entinostat plus imiquimod combination treatment markedly reduced BCC counts associated with increased CD8+ T cell infiltration in Ptchl +/- skin compared with entinostat, imiquimod, and vehicle control treatments (FIGs. 3L-3M, and 5D-5E).

Example 5: Primary human BCC’s low immunogenicity is linked to low IRF1 and high Foxcl expression

To extend our findings to humans, we evaluated IRF1 and Foxcl expression in primary human BCC versus SCC. Markedly fewer BCC cancer cells expressed IRF1 compared with SCC (FIGs. 4A-4B). While absent in SCC, Foxcl was highly expressed by BCC cancer cells, especially in the center of tumor foci (FIG. 4C). Interestingly, Foxcl+ BCC cancer cells lacked Ki67 expression, reminiscent of quiescent hair follicle stem cells (FIGs. 6A-6B)³³. BCC lacked HLA-I^hlgh expressing cells found in SCC; however, a subset of BCC cancer cells expressed medium HLA-I levels (HLA-I^mcdllim) while other cells lacked detectable HLA-I expression (HLA-I" ^/LOW) (FIG 4C). Significantly fewer Foxcl+ BCC cancer cells expressed HLA-I^mcdllim compared with Foxcl- cancer cells (FIG. 4D). scRNA-Seq analysis showed that Foxcl+ BCC cancer cells had lower APM gene expression compared with Foxcl - cancer cells (FIG. 6C). To elucidate the spatial relationship between Foxcl and HLA- I expression, we measured the distance between Foxcl+ cells and HLA-I^mcdll,m versus HLA-T^/low cells in BCC. HLA-I^mcdllim cancer cells were located significantly farther away from Foxcl+ cells compared with HLA-I"^/low cancer cells (FIGs. 4E-4F). These results indicate Foxcl -mediated HLA-I downregulation via epigenetic modification, which can persist in the progenitor cells over several mitoses and govern BCC’s low immunogenicity (FIG. 4G)⁵⁸.

To determine whether disparate BCC and SCC immunogenicity is intrinsic to their development and not secondary to age-associated mutations in the skin⁵⁹, we examined pediatric BCC and SCC cases. Among 124 pediatric patients with KC⁶⁰, 21 were identified with iatrogenic risk factors associated with their KC. Comparing iatrogenic risk factors for SCC versus BCC revealed that all pediatric patients with SCC (8 of 8) had prolonged immunosuppression as a risk factor. By contrast, 69% of the pediatric patients with BCC (9 of 13) had undergone chemotherapy and/or radiation therapy without immunosuppression (FIG. 6D). This aligns with a recent study showing that radiotherapy is the dominant risk factor for subsequent BCC development in childhood cancer survivors⁶¹. This clinical observation suggests that immune surveillance is critical for SCC but not BCC prevention in the pediatric population. Under immunosuppressive drug therapy, minimal CD8+ T cells infiltrated pediatric SCCs, which was not significantly different from CD8+ T cell infiltrate in pediatric BCCs (FIGs. 4H-4I). Like older adults, Foxcl expression was only detectable in pediatric BCC, and HLA-I expression was significantly lower in pediatric BCC compared with SCC (FIGs. 4J-4K). These findings substantiate the role of Foxcl as a critical transcription factor in BCC development from hair follicle and interfollicular epidermis stem cells, which suppresses HLA-I expression, leading to BCC’s low immunogenicity. REFERENCES

1. Albert, M.R., and Weinstock, M.A. (2003). Keratinocyte carcinoma. CA Cancer J. Clin. 53, 292-302. 10.3322/canjclin.53.5.292.

2. Madan, V., Lear, J.T., and Szeimies, R.M. (2010). Non-melanoma skin cancer. Lancet 375, 673-685. 10.1016/80140-6736(09)61196-X.

3. Rogers, H.W., Weinstock, M.A., Feldman, S.R., and Coldiron, B.M. (2015). Incidence Estimate of Nonmelanoma Skin Cancer (Keratinocyte Carcinomas) in the U.S. Population, 2012. JAMA dermatology 151, 1081-1086. 10.1001/jamadermatol.2015.1187.

4. Dika, E., Scarfi, F., Ferracin, M., Broseghini, E., Marcelli, E., Bortolani, B., Campione, E., Riefolo, M., Ricci, C., and Lambertini, M. (2020). Basal Cell Carcinoma: A Comprehensive Review. Int. J. Mol. Sci. 21. 10.3390/ijms21155572.

5. Goodman, A.M., Kato, S., Cohen, P.R., Boichard, A., Frampton, G., Miller, V., Stephens, P.J., Daniels, G.A., and Kurzrock, R. (2018). Genomic landscape of advanced basal cell carcinoma: Implications for precision treatment with targeted and immune therapies. Oncoimmunology 7, e 1404217.

10.1080/2162402X.2017.1404217.

6. Chalmers, Z.R., Connelly, C.F., Fabrizio, D., Gay, L., Ali, S.M., Ennis, R., Schrock,

A., Campbell, B., Shlien, A., Chmielecki, J., et al. (2017). Analysis of 100,000 human cancer genomes reveals the landscape of tumor mutational burden. Genome Med. 9, 34. 10.1186/S13073-017-0424-2.

7. Yarchoan, M., Hopkins, A., and Jaffee, E.M. (2017). Tumor Mutational Burden and Response Rate to PD-1 Inhibition. N. Engl. J. Med. 377, 2500-2501. 10.1056/NEJMC1713444.

8. Rischin, D., Migden, M.R., Lim, A.M., Schmults, C.D., Khushalani, N.I., Hughes,

B.G.M., Schadendorf, D., Dunn, L.A., Hernandez-Aya, L., Chang, A.L.S., et al. (2020). Phase 2 study of cemiplimab in patients with metastatic cutaneous squamous cell carcinoma: primary analysis of fixed-dosing, long-term outcome of weight-based dosing. J Immunother Cancer 8. 10. 1136/jitc-2020-000775.

9. Shalhout, S.Z., Emerick, K.S., Kaufman, H.L., and Miller, D.M. (2021). Immunotherapy for Non-melanoma Skin Cancer. Curr. Oncol. Rep. 23, 125. 10.1007/sl 1912-021-01120-z.

10. Hartevelt, M.M., Bavinck, J.N., Kootte, A.M., Vermeer, B.J., and Vandenbroucke, J.P. (1990). Incidence of skin cancer after renal transplantation in The Netherlands. Transplantation 49, 506-509. 10.1097/00007890-199003000-00006.

11. Lindelof, B., Sigurgeirsson, B., Gabel, H., and Stem, R.S. (2000). Incidence of skin cancer in 5356 patients following organ transplantation. Br. J. Dermatol. 143, 513- 519.

12. Jensen, P., Hansen, S., Moller, B., Leivestad, T., Pfeffer, P., Geiran, O., Fauchald, P., and Simonsen, S. (1999). Skin cancer in kidney and heart transplant recipients and different long-term immunosuppressive therapy regimens. J. Am. Acad. Dermatol.

40, 177-186. 10.1016/s0190-9622(99)70185-4.

13. Harwood, C.A., Proby, C.M., McGregor, J.M., Sheaff, M.T., Leigh, I.M., and Cerio, R. (2006). Clinicopathologic features of skin cancer in organ transplant recipients: a retrospective case-control series. J. Am. Acad. Dermatol. 54, 290-300. 10.1016/j.jaad.2005. 10.049.

14. Moloney, F.J., Comber, H., Conlon, P.J., and Murphy, G.M. (2006). The role of immunosuppression in the pathogenesis of basal cell carcinoma. Br. J. Dermatol. 154, 790-791. 10. 111 l/j,1365-2133.2006.07156.x.

15. Tran, H., Chen, K., and Shumack, S. (2003). Epidemiology and aetiology of basal cell carcinoma. Br. J. Dermatol. 149 Suppl 66, 50-52. 10. 1046/j .0366- 077x.2003.05622.x. 16. Krynitz, B., Olsson, H., Lundh Rozell, B., Lindelof, B., Edgren, G., and Smedby, K.E. (2016). Risk of basal cell carcinoma in Swedish organ transplant recipients: a population based study. Br. J. Dermatol. 174, 95-103. 10.1111/bjd. 14153.

17. Tiwari, A., Oravecz, T., Dillon, L.A., Italiano, A., Audoly, L., Fridman, W.H., and Clifton, G.T. (2023). Towards a consensus definition of immune exclusion in cancer. Front. Immunol. 14, 1084887. 10.3389/fimmu.2023.1084887.

18. Ogino, T., Shigyo, H., Ishii, H., Katayama, A., Miyokawa, N., Harabuchi, Y ., and Ferrone, (2006). HLA class I antigen down-regulation in primary laryngeal squamous cell carcinoma lesions as a poor prognostic marker. Cancer Res. 66, 9281-9289.

10.1158/0008-5472. CAN-06-0488.

19. Dhatchinamoorthy, K., Colbert, J.D., and Rock, K.L. (2021). Cancer Immune Evasion Through Loss of MHC Class I Antigen Presentation. Front. Immunol. 12, 636568. 10.3389/fimmu.2021.636568.

20. Jhunjhunwala, S., Hammer, C., and Delamarre, L. (2021). Antigen presentation in cancer: insights into tumour immunogenicity and immune evasion. Nat. Rev. Cancer 21, 298-312. 10.1038/s41568-021-00339-z.

21. Galon, J., Pages, F., Marincola, F.M., Thurin, M., Trinchieri, G., Fox, B.A., Gajewski, T.F., and Ascierto, P.A. (2012). The immune score as a new possible approach for the classification of cancer. J. Transl. Med. 10, 1. 10. 1186/1479-5876- 10-1.

22. Cornel, A.M., Mimpen, I.L., and Nierkens, S. (2020). MHC Class I Downregulation in Cancer: Underlying Mechanisms and Potential Targets for Cancer Immunotherapy. Cancers (Basel) 12. 10.3390/cancersl2071760.

23. Noubissi, F.K., Kim, T., Kawahara, T.N., Aughenbaugh, W.D., Berg, E., Longley, B.J., Athar, M., and Spiegelman, V.S. (2014). Role of CRD-BP in the growth of human basal cell carcinoma cells. J. Invest. Dermatol. 134, 1718-1724. 10.1038/jid.2014.17.

24. Rheinwald, J.G., and Beckett, M.A. (1981). Tumorigenic keratinocyte lines requiring anchorage and fibroblast support cultured from human squamous cell carcinomas. Cancer Res. 41, 1657-1663.

25. Al Labban, D., Jo, S.H., Ostano, P., Saglietti, C., Bongiovanni, M., Panizzon, R., and Dotto, G.P. (2018). Notch-effector CSL promotes squamous cell carcinoma by repressing histone demethylase KDM6B. J. Clin. Invest. 128, 2581-2599.

10.1172/JCI96915.

26. Boukamp, P., Petrussevska, R.T., Breitkreutz, D., Hornung, J., Markham, A., and Fusenig, N.E. (1988). Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J. Cell Biol. 106, 761-771.

10.1083/jcb.106.3.761.

27. Recipon, M., Agniel, R., Leroy-Dudal, J., Fritz, T., Carreiras, F., Hermitte, F., Hubac, S., Gallet, O., and Kellouche, S. (2023). Targeting cell-derived markers to improve the detection of invisible biological traces for the purpose of genetic-based criminal identification. Sci. Rep. 13, 18105. 10.1038/s41598-023-45366-y.

28. Johnson, R.L., Rothman, A.L., Xie, J., Goodrich, L.V., Bare, J.W., Bonifas, J.M., Quinn, A.G., Myers, R.M., Cox, D.R., Epstein, E.H., Jr., and Scott, M.P. (1996). Human homolog of patched, a candidate gene for the basal cell nevus syndrome. Science 272, 1668-1671. 10.1126/science.272.5268.1668.

29. Hahn, H., Wicking, C., Zaphiropoulous, P.G., Gailani, M.R., Shanley, S., Chidambaram, A., Vorechovsky, I., Holmberg, E., Unden, A.B., Gillies, S., et al. (1996). Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome. Cell 85, 841-851. 10.1016/s0092-8674(00)81268-4.

30. Otsuka, A., Dreier, J., Cheng, P.F., Nageli, M., Lehmann, H., Felderer, L., Frew, I.J., Matsushita, S., Levesque, M.P., and Dummer, R. (2015). Hedgehog pathway inhibitors promote adaptive immune responses in basal cell carcinoma. Clin Cancer Res 21, 1289- 1297. 10.1158/1078-0432.CCR-14-2110. 31. Lorenzi, S., Forloni, M., Cifaldi, L., Antonucci, C., Citti, A., Boldrini, R., Pezzullo, M., Castellano, A., Russo, V., van der Bruggen, P., et al. (2012). IRF1 and NF-kB restore MHC class I-restricted tumor antigen processing and presentation to cytotoxic T cells in aggressive neuroblastoma. PLoS One 7, e46928.

10.1371/joumal.pone.0046928.

32. Vijayan, S., Sidiq, T., Yousuf, S., van den Eisen, P.J., and Kobayashi, K.S. (2019). Class I transactivator, NLRC5 : a central player in the MHC class I pathway and cancer immune surveillance. Immunogenetics 71, 273-282. 10.1007/s00251-019- 01106-z.

33. Agudo, J., Park, E.S., Rose, S.A., Alibo, E., Sweeney, R., Dhainaut, M., Kobayashi, K.S., Sachidanandam, R., Baccarini, A., Merad, M., and Brown, B.D. (2018). Quiescent Tissue Stem Cells Evade Immune Surveillance. Immunity 48, 271-285 e275. 10.1016/j.immuni.2018.02.001.

34. Lay, K., Kume, T., and Fuchs, E. (2016). FOXCI maintains the hair follicle stem cell niche and governs stem cell quiescence to preserve long-term tissue-regenerating potential. Proc. Natl. Acad. Sci. U. S. A. 113, E1506-1515.

10.1073/pnas. 1601569113.

35. Wang, L., Siegenthaler, J.A., Dowell, R.D., and Yi, R. (2016). Foxcl reinforces quiescence in self-renewing hair follicle stem cells. Science 351, 613-617.

10.1126/science.aad5440.

36. Grachtchouk, M., Pero, J., Yang, S.H., Ermilov, A.N., Michael, L.E., Wang, A., Wilbert, D., Patel, R.M., Ferris, J., Diener, J., et al. (2011). Basal cell carcinomas in mice arise from hair follicle stem cells and multiple epithelial progenitor populations. J. Clin. Invest. 121, 1768-1781. 10.1172/JCI46307.

37. Wang, G.Y., Wang, J., Mancianti, M.L., and Epstein, E.H., Jr. (2011). Basal cell carcinomas arise from hair follicle stem cells in Ptchl(+/-) mice. Cancer Cell 19, 114- 124. 10.1016/j.ccr.2010.11.007.

38. Lalmansingh, A.S., Karmakar, S., Jin, Y ., and Nagaich, A.K. (2012). Multiple modes of chromatin remodeling by Forkhead box proteins. Biochim Biophys Acta 1819, 707-715. 10.1016/j.bbagrm.2012.02.018.

39. Chen, X., Wei, H., Li, J., Liang, X., Dai, S., Jiang, L., Guo, M., Qu, L., Chen, Z., Chen, L., and Chen, Y. (2019). Structural basis for DNA recognition by FOXC2. Nucleic Acids Res. 47, 3752-3764. 10.1093/nar/gkz077.

40. 40. He, B., Ebarasi, L., Zhao, Z., Guo, J., Ojala, J.R., Hultenby, K., De Vai, S., Betsholtz, C.,and Tryggvason, K. (2014). Lmxlb and FoxC combinatorially regulate podocin expression in podocytes. J. Am. Soc. Nephrol. 25, 2764-2777.

10.1681/ASN.2012080823.

41. Xue, H„ Liu, F„ Ai, Z„ Ke, J., Yu, M„ Chen, B„ and Guo, Z. (2021). FOXCI Downregulates Nanog Expression by Recruiting HDAC2 to Its Promoter in F9 Cells Treated by Retinoic Acid. Int. J. Mol. Sci. 22. 10.3390/ijms22052255.

42. Simeoni, F., and Somervaille, T.C. (2021). Enhancer recruitment of a RUNX1, HDAC1 and TLE3 co-repressor complex by mis-expressed FOXCI blocks differentiation in acute myeloid leukemia. Mol Cell Oncol 8, 2003161. 10.1080/23723556.2021.2003161.

43. Lam, E.W., Brosens, J. J., Gomes, A.R., and Koo, C.Y. (2013). Forkhead box proteins: tuning forks for transcriptional harmony. Nat. Rev. Cancer 13, 482-495. 10.1038/nrc3539.

44. Litvak, V., Ratushny, A.V., Lampano, A.E., Schmitz, F., Huang, A.C., Raman, A., Rust, A.G., Bergthaler, A., Aitchison, J.D., and Aderem, A. (2012). A FOXO3-IRF7 gene regulatory circuit limits inflammatory sequelae of antiviral responses. Nature 490, 421-425. 10.1038/naturel l428.

45. Lee, B.I., Park, S.H., Kim, J.W., Sausville, E.A., Kim, H.T., Nakanishi, O., Trepel, J.B., and Kim, S.J. (2001). MS-275, a histone deacetylase inhibitor, selectively induces transforming growth factor beta type II receptor expression in human breast cancer cells. Cancer Res. 61, 931-934.

46. Aszterbaum, M., Epstein, J., Anthony, O., Douglas, V., Leboit, P.E., Scott, M.P., and Epstein, E.H. (1999). Ultraviolet and ionizing radiation enhance the growth of BCCs and trichoblastomas in patched heterozygous knockout mice. Nat. Med. 5, 1285- 1291. 10.1038/15242.

47. Youssef, K.K., Van Keymeulen, A., Lapouge, G., Beck, B., Michaux, C., Achouri, Y ., Sotiropoulou, P.A., and Blanpain, C. (2010). Identification of the cell lineage at the origin of basal cell carcinoma. Nature cell biology 12, 299-305. 10.1038/ncb2031.

48. Tan, S.T., Ghaznawie, M., Heenan, P.J., and Dosan, R. (2018). Basal Cell Carcinoma Arises from Interfollicular Layer of Epidermis. J. Oncol. 2018, 3098940. 10.1155/2018/3098940.

49. Bansaccal, N., Vieugue, P., Sarate, R., Song, Y ., Minguijon, E., Miroshnikova, Y.A., Zeuschner, D., Collin, A., Allard, J., Engelman, D., et al. (2023). The extracellular matrix dictates regional competence for tumour initiation. Nature 623, 828-835. 10.1038/s41586-023-06740-y.

50. Youssef, K.K., Lapouge, G., Bouvree, K., Rorive, S., Brohee, S., Appelstein, O., Larsimont, J.C., Sukumaran, V., Van de Sande, B., Pucci, D., et al. (2012). Adult interfollicular tumour-initiating cells are reprogrammed into an embryonic hair follicle progenitor-like fate during basal cell carcinoma initiation. Nat. Cell Biol. 14, 1282-1294. 10.1038/ncb2628.

51. Zhu, X., Leboeuf, M., Liu, F., Grachtchouk, M., Seykora, J.T., Morrisey, E.E., Dlugosz, A.A., and Millar, S.E. (2022). HDAC1/2 Control Proliferation and Survival in Adult Epidermis and Pre-Basal Cell Carcinoma through pl6 and p53. J. Invest. Dermatol. 142, 77-87 elO. 10.1016/j .jid.2021.05.026.

52. Garcia-de-la-Fuente, M.R., Santacana, M., Valls, J., Vilardell, F., Fernandez Armenteros, J.M., Pujol, R., Gari, E., and Casanova, J.M. (2018). Cytokeratin Profde of Basal Cell Carcinomas According to the Degree of Sun Exposure and to the Anatomical Localization. Am. J. Dermatopathol. 40, 342-348.

10.1097/D AD.0000000000001042.

53. Anderson-Dockter, H., Clark, T., Iwamoto, S., Lu, M., Fiore, D., Falanga, J.K., and Falanga, V. (2012). Diagnostic utility of cytokeratin 17 immunostaining in morpheaform basal cell carcinoma and for facilitating the detection of tumor cells at the surgical margins. Dermatol. Surg. 38, 1357-1366. 10.1111/j .1524- 4725.2012.02417.x.

54. Geisse, J., Caro, I., Lindholm, J., Golitz, L., Stampone, P., and Owens, M. (2004). Imiquimod 5% cream for the treatment of superficial basal cell carcinoma: results from two phase III, randomized, vehicle -controlled studies. J. Am. Acad. Dermatol. 50, 722-733. 10.1016/j.jaad.2003. 11.066.

55. Bukowinski, A., Chang, B., Reid, J.M., Liu, X., Minard, C.G., Trepel, J.B., Lee, M.J., Fox, E., and Weigel, B.J. (2021). A phase 1 study of entinostat in children and adolescents with recurrent or refractory solid tumors, including CNS tumors: Trial ADVL1513, Pediatric Early Phase -Clinical Trial Network (PEP-CTN). Pediatr. Blood Cancer 68, e28892. 10.1002/pbc.28892.

56. Pili, R., Quinn, D.I., Hammers, H.J., Monk, P., George, S., Dorff, T.B., Olencki, T., Shen, L., Orillion, A., Lamonica, D., et al. (2017). Immunomodulation by Entinostat in Renal Cell Carcinoma Patients Receiving High-Dose Interleukin 2: A Multicenter, Single-Arm, Phase Eli Trial (NCI-CTEP#7870). Clin Cancer Res 23, 7199-7208. 10.1158/1078-0432. CCR-17-1178.

57. Yeruva, S.L.H., Zhao, F., Miller, K.D., Tevaarwerk, A.J., Wagner, L.I., Gray, R.J., Sparano, J.A., and Connolly, R.M. (2018). E2112: randomized phase iii trial of endocrine therapy plus entinostat/placebo in patients with hormone receptor-positive advanced breast cancer. NPJ Breast Cancer 4, 1. 10.1038/s41523-017-0053-3. 58. Halsall, J.A., Andrews, S., Krueger, F., Rutledge, C.E., Ficz, G., Reik, W., and Turner, B.M. (2021). Histone modifications form a cell-type-specific chromosomal bar code that persists through the cell cycle. Sci. Rep. 77, 3009. 10.1038/s41598-021- 82539-z.

59. Hernando, B., Dietzen, M., Parra, G., Gil-Barrachina, M., Pitarch, G., Mahiques, L., Valcuende-Cavero, F., McGranahan, N., and Martinez-Cadenas, C. (2021). The effect of age on the acquisition and selection of cancer driver mutations in sun -exposed normal skin. Ann. Oncol. 32, 412-421. 10.1016/j.annonc.2020.11.023.

60. Huang, J.T., Coughlin, C.C., Hawryluk, E.B., Hook, K., Humphrey, S.R., Kruse, L., Lawley, L., Al-Sayegh, H., London, W.B., Marghoob, A., et al. (2019). Risk Factors and Outcomes of Nonmelanoma Skin Cancer in Children and Young Adults. J. Pediatr. 277, 152-158. 10. 1016/j.jpeds.2019.04.017.

61. Gibson, T.M., Karyadi, D.M., Hartley, S.W., Arnold, M.A., Berrington de Gonzalez, A., Conces, M.R., Howell, R.M., Kapoor, V., Leisenring, W.M., Neglia, J.P., et al. (2024). Polygenic risk scores, radiation treatment exposures and subsequent cancer risk in childhood cancer survivors. Nat. Med. 10.1038/s41591-024-02837-7.

62. Walter, A., Barysch, M.J., Behnke, S., Dziunycz, P., Schmid, B., Ritter, E., Gnjatic, S., Kristiansen, G., Moch, H., Knuth, A., et al. (2010). Cancer-testis antigens and immunosurveillance in human cutaneous squamous cell and basal cell carcinomas. Clin Cancer Res 76, 3562-3570. 10.1158/1078-0432.CCR-09-3136.

63. Hua, L.A., Kagen, C.N., Carpenter, R.J., and Goltz, R.W. (1985). HLA and beta 2- microglobulin expression in basal and squamous cell carcinomas of the skin. Int. J. Dermatol. 24, 660-663. 10.111 l/j,1365-4362.1985.tb05719.x.

64. Gong, X., and Karchin, R. (2022). Pan-Cancer HLA Gene-Mediated Tumor Immunogenicity and Immune Evasion. Mol. Cancer Res. 20, 1272-1283. 1158/1541- 7786.MCR-21-0886.

65. Quist, S.R., Eckardt, M., Kriesche, A., and Gollnick, H.P. (2016). Expression of epidermal stem cell markers in skin and adnexal malignancies. Br. J. Dermatol. 775, 520-530. 10.1111/bjd. 14494.

66. Eberl, M., Mangelberger, D., Swanson, J.B., Verhaegen, M.E., Harms, P.W., Frohm, M.L., Dlugosz, A.A., and Wong, S.Y. (2018). Tumor Architecture and Notch Signaling Modulate Drug Response in Basal Cell Carcinoma. Cancer Cell 33, 229- 243 e224. 10. 1016/j .ccell.2017. 12.015.

67. Trieu, K.G., Tsai, S.Y., Eberl, M., Ju, V., Ford, N.C., Doane, O.J., Peterson, J.K., Veniaminova, N.A., Grachtchouk, M., Harms, P.W., et al. (2022). Basal cell carcinomas acquire secondary mutations to overcome dormancy and progress from microscopic to macroscopic disease. Cell Rep. 39, 110779.

10.1016/j.celrep.2O22.110779.

68. Sellheyer, K. (2011). Basal cell carcinoma: cell of origin, cancer stem cell hypothesis and stem cell markers. Br. J. Dermatol. 164, 696-711. 10.111 l/j.1365 2133.2010.10158.x.

69. Krahl, D., and Sellheyer, K. (2010). Basal cell carcinoma and pilomatrixoma mirror human follicular embryogenesis as reflected by their differential expression patterns of SOX9 and beta-catenin. Br. J. Dermatol. 162, 1294-1301. 10. 1111/j .1365- 2133.2010.09630.x.

70. Kruger, K., Blume-Peytavi, U., and Orfanos, C.E. (1999). Basal cell carcinoma possibly originates from the outer root sheath and/or the bulge region of the vellus hair follicle. Arch. Dermatol. Res. 297, 253-259. 10.1007/s004030050405.

71. Asada, M., Schaart, F.M., de Almeida, H.L., Jr., Korge, B., Kurokawa, I., Asada, Y., and Orfanos, C.E. (1993). Solid basal cell epithelioma (BCE) possibly originates from the outer root sheath of the hair follicle. Acta Derm. Venereol. 73, 286-292.

72. Peterson, S.C., Eberl, M., Vagnozzi, A.N., Belkadi, A., Veniaminova, N.A., Verhaegen, M.E., Bichakjian, C.K., Ward, N.L., Dlugosz, A.A., and Wong, S.Y. (2015). Basal cell carcinoma preferentially arises from stem cells within hair follicle and mechanosensory niches. Cell Stem Cell 16, 400-412. 10.1016/j.stem.2015.02.006.

73. Ancel, S., Stuelsatz, P., and Feige, J.N. (2021). Muscle Stem Cell Quiescence: Controlling Sternness by Staying Asleep. Trends Cell Biol. 31, 556-568. 10.1016/j.tcb.2021.02.006.

74. Sistigu, A., Musella, M., Galassi, C., Vitale, I., and De Maria, R. (2020). Tuning Cancer Fate: Tumor Microenvironment's Role in Cancer Stem Cell Quiescence and Reawakening. Front. Immunol. 11, 2166. 10.3389/fhnmu.2020.02166.

75. Chen, W., Dong, J., Haiech, J., Kilhoffer, M.C., and Zeniou, M. (2016). Cancer Stem Cell Quiescence and Plasticity as Major Challenges in Cancer Therapy. Stem Cells Int. 2016, 1740936. 10.1155/2016/1740936.

76. Rodems, T.S., Heninger, E., Stahlfeld, C.N., Gilsdorf, C.S., Carlson, K.N., Kircher, M.R., Singh, A., Krueger, T.E.G., Beebe, D.J., Jarrard, D.F., et al. (2022). Reversible epigenetic alterations regulate class I HLA loss in prostate cancer. Commun Biol 5, 897. 10.1038/s42003-022-03843-6.

77. Chen, X., Pan, X., Zhang, W., Guo, H., Cheng, S., He, Q., Yang, B., and Ding, L. (2020). Epigenetic strategies synergize with PD-L1/PD-1 targeted cancer immunotherapies to enhance antitumor responses. Acta Pharm Sin B 10, 723-733. 10.1016/j.apsb.2019.09.006.

78. Gomez, S., Tabemacki, T., Kobyra, J., Roberts, P., and Chiappinelli, K.B. (2020). Combining epigenetic and immune therapy to overcome cancer resistance. Semin. Cancer Biol. 65, 99-113. 10.1016/j.semcancer.2019.12.019.

79. Rosenberg, A.R., Tabacchi, M., Ngo, K.H., Wallendorf, M., Rosman, I.S., Cornelius, L.A., and Demehri, S. (2019). Skin cancer precursor immunotherapy for squamous cell carcinoma prevention. JCI Insight 4. 10. 1172/j ci. insight. 125476.

80. Stratigos, A. J., Sekulic, A., Peris, K., Bechter, O., Prey, S., Kaatz, M., Lewis, K.D., Basset-Seguin, N., Chang, A.L.S., Dalle, S., et al. (2021). Cemiplimab in locally advanced basal cell carcinoma after hedgehog inhibitor therapy: an open-label, multicentre, single-arm, phase 2 trial. Lancet Oncol. 22, 848-857. 10.1016/S 1470- 2045(21)00126-1.

81. Stanley, M.A. (2002). hniquimod and the imidazoquinolones: mechanism of action and therapeutic potential. Clin Exp Dermatol 27, 571-577.

10.1046/j.13652230.2002.01151.x.

82. Urosevic, M., Maier, T., Benninghoff, B., Slade, H., Burg, G., and Dummer, R. (2003). Mechanisms underlying imiquimod-induced regression of basal cell carcinoma in vivo. Arch. Dermatol. 139, 1325-1332. 10.1001/archderm.l39.10.1325.

83. Swain, N., Tripathy, A., Padhan, P., Raghav, S.K., and Gupta, B. (2022). Toll-like receptor-7 activation in CD8+ T cells modulates inflammatory mediators in patients with rheumatoid arthritis. Rheumatol. Int. 42, 1235-1245. 10.1007/s00296-021- 05050-8.

84. Tilley, C., Deep, G., Agarwal, C., Wempe, M.F., Biedermann, D., Valentova, K., Kren, V., and Agarwal, R. (2016). Silibinin and its 2,3 -dehydro-derivative inhibit basal cell carcinoma growth via suppression of mitogenic signaling and transcription factors activation. Mol. Carcinog. 55, 3-14. 10. 1002/mc.22253.

85. 85. So, P.L., Langston, A.W., Daniallinia, N., Hebert, J.L., Fujimoto, M.A., Khaimskiy, Y., Aszterbaum, M., and Epstein, E.H., Jr. (2006). Long-term establishment, characterization and manipulation of cell lines from mouse basal cell carcinoma tumors. Exp. Dermatol. 15, 742-750. 10.1111/j.1600-0625.2006.00465.x.

86. Lucena, S.R., Zamarron, A., Carrasco, E., Marigil, M.A., Mascaraque, M., Fernandez-Guarino, M., Gilaberte, Y ., Gonzalez, S., and Juarranz, A. (2019). Characterisation of resistance mechanisms developed by basal cell carcinoma cells in response to repeated cycles of Photodynamic Therapy. Sci. Rep. 9, 4835.

10.1038/s41598-019-41313-y. 87. Yao, C.D., Haensel, D., Gaddam, S., Patel, T., Atwood, S.X., Sarin, K.Y., Whitson, R.J., McKellar, S., Shankar, G., Aasi, S., et al. (2020). AP-1 and TGFss cooperativity drives non-canonical Hedgehog signaling in resistant basal cell carcinoma. Nat Commun / /. 5079. 10.1038/s41467-020-18762-5.

88. Guerrero-Juarez, C.F., Lee, G.H., Liu, Y., Wang, S., Karikomi, M., Sha, Y., Chow, R.Y., Nguyen, T.T.L., Iglesias, V.S., Aasi, S., et al. (2022). Single-cell analysis of human basal cell carcinoma reveals novel regulators of tumor growth and the tumor microenvironment. Sci Adv 8, eabm7981. 10.1126/sciadv.abm7981.

89. Zou, D.D., Sun, Y.Z., Li, X.J., Wu, W.J., Xu, D., He, Y.T., Qi, J., Tu, Y ., Tang, Y ., Tu, Y.H., et al. (2023). Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma. Elife 12.

10.7554/eLife.85270.

90. Satija, R., Farrell, J.A., Gennert, D., Schier, A.F., and Regev, A. (2015). Spatial reconstruction of single-cell gene expression data. Nat. Biotechnol. 33, 495-502. 10.1038/nbt.3192.

91. Colzani, M., Waridel, P., Laurent, J., Faes, E., Ruegg, C., and Quadroni, M. (2009). Metabolic labeling and protein linearization technology allow the study of proteins secreted by cultured cells in serum-containing media. J. Proteome Res. 8, 4779-4788. 10.1021/pr900476b.

92. Goodrich, L.V., Milenkovic, L., Higgins, K.M., and Scott, M.P. (1997). Altered neural cell fates and medulloblastoma in mouse patched mutants. Science 277, 1109- 1113. 10.1126/science.277.5329. 1109.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating or preventing basal cell carcinoma in a subject comprising administering to the subject an effective amount of an HD AC inhibitor in combination with an effective amount of one or more of a TLR7/8 agonist, vitamin D analog, or 5-FU.

2. A method of treating of preventing basal cell carcinoma in a subject comprising administering to the subject an effective amount of an HD AC inhibitor, a systemic immunotherapy, and one or more of a TLR7/8 agonist, vitamin D analog, or 5-FU.

3. A method of treating or preventing non-immunogenic skin cancers comprising administering to the subject an effective amount of an HD AC inhibitor, in combination with an effective amount of a TLR7/8 agonist, vitamin D analog, or 5-FU.

4. A method of treating or preventing a Foxcl positive tumor comprising administering to the subject an effective amount of an HD AC inhibitor in combination with an effective amount of one or more of a TLR7/8 agonist, vitamin D analog, or 5-FU.

5. The method of claim 4, wherein the Foxcl positive tumor is a tumor that comprises one or more Foxcl positive cells.

6. The method of any of the above claims, wherein the HD AC inhibitor comprises panobinostat, abexinostat, entinostat, mocetinostat, vorinostat, pracinostat, ricolinostat, apicidin, fimepinostat, P300 histone acetylatransferase, tubastatin A, tucidinostat, romidepsin, belinostat, givinostat, or valproic acid.

7. The method of any of the above claims, wherein the HD AC inhibitor comprises entinostat.

8. The method of any of the above claims, wherein the HD AC inhibitor is administered topically or systemically.

9. The method of any of the above claims, wherein the systemic immunotherapy is an immune checkpoint inhibitor.

10. The method of any of the above claims, wherein the systemic immunotherapy is a sonic hedgehog pathway inhibitor selected from vismodegib and sonidegib.

11. The method of any of the above claims, wherein the TLR7/8 agonist is selected from imiquimod, resiquimod, 852-A, vesatolimod, AZD8848, motolimod, and selgantolimod.

12. The method of any of the above claims, wherein the TLR7/8 agonist is imiquimod.

13. The method of any of the above claims, wherein the non-immunogenic skin cancer comprises Extramammary Paget’s disease, chest wall metastasis of breast cancer, malignant melanoma, or adnexal carcinoma.

14. The method of any of the above claims, wherein the vitamin D analog comprises doxercalciferol, paricalcitol, calcitriol, tacalcitol, alfacalcidol, calcijex, calcipotriol, falecalcitriol, vitamin D, dihydroxycholecalciferol, rayaldee, rocaltrol, calcifediol, cholecalciferol, decara, oleovitamin d3, or trionex.

15. The method of any of the above claims, wherein the vitamin D analog comprises calcipotriol.

16. The method of any of the above claims, wherein the HD AC inhibitor is administered separately from the one or more of a TLR7/8 agonist, vitamin D analog, or 5-FU.

17. The method of any of the above claims, wherein the HD AC inhibitor is administered concomitantly or sequentially.

18. The method of any of the above claims, wherein the HD AC inhibitor is administered together with the one or more of a TLR7/8 agonist, vitamin D analog, or 5-FU in the same composition.

19. The method of any of the above claims, wherein the administering comprises administering the composition(s) to a tumor.