WO2025068368A1

WO2025068368A1 - Inhibitors for treating cancer

Info

Publication number: WO2025068368A1
Application number: PCT/EP2024/077056
Authority: WO
Inventors: Eleonora LEUCCI; Elisabetta GROAZ; Tiziana Bonaldi; Alessandro CUOMO
Original assignee: Katholieke Universiteit Leuven; Universita degli Studi di Milano; Istituto Europeo di Oncologia SRL IEO
Current assignee: Katholieke Universiteit Leuven; Universita degli Studi di Milano; Istituto Europeo di Oncologia SRL IEO
Priority date: 2023-09-26
Filing date: 2024-09-26
Publication date: 2025-04-03
Anticipated expiration: 2026-03-26

Abstract

The present invention relates to inhibitors of TMED2-DT expression, in particular inhibitors of TMED2-DT expression selected from the group consisting of antisense oligonucleotide (ASO), RNAi agent and ribozyme. The invention further relates to use of TMED2-DT expression inhibitors for the treatment of cancer.

Description

INHIBITORS FOR TREATING CANCER

FIELD OF THE INVENTION

The present invention relates to inhibitors of TMED2-DT expression and their use in the treatment of cancer, in particular in the treatment of melanoma.

BACKGROUND OF THE INVENTION

The introduction of immune checkpoint inhibitors has revolutionized cancer treatment. However not all cancer types respond equally well and moreover toxicities and intrinsic and acquired resistance mechanisms hamper the success of immune therapy in a significant portion of patients. There is thus a need for additional treatments that can overcome resistance to therapy and avoid toxicities.

Here the Inventors have found that inhibition of TMED2-DT expression increases melanoma cell killing by immune cells in vitro and that expression of TMED2-DT correlates with poor patient survival. Accordingly, the inhibitors of the present invention are valuable alternatives to immune checkpoint inhibitors in the treatment of cancer, or may be combined with such immune checkpoint inhibitors to even further increase the efficacy of the cancer treatment.

SUMMARY

The present invention relates to an inhibitor of TMED2-DT expression. In one embodiment, the inhibitor of TMED2-DT expression is a specific inhibitor.

In one embodiment, the inhibitor of TMED2-DT expression is selected from the group consisting of antisense oligonucleotide (ASO), RNAi agent and ribozyme.

In one embodiment, the ASO, RNAi agent or ribozyme comprises a nucleotide sequence complementary to a TMED2-DT transcript. In one embodiment, the inhibitor of TMED2-DT expression is an ASO inducing degradation of a TMED2-DT transcript, preferably inducing RNAse H-mediated degradation of a TMED2-DT transcript.

In one embodiment, the inhibitor of TMED2-DT expression is a translation-blocking ASO, preferably a translation-blocking ASO preventing translation of the protein of amino acid sequence SEQ ID NO. 1.

In one embodiment, the RNAi agent is selected from the group consisting of siRNA, shRNA, dsRNA and nucleic acid coding for a dsRNA or for a shRNA.

In one embodiment, the inhibitor of TMED2-DT expression is an ASO having an overall nucleotide sequence length of at least 10 nucleotides, preferably of at least 11, 12, 13, 14, 15, 16, 17 or 18 nucleotides, more preferably of at least 19 or 20 nucleotides.

In one embodiment, the inhibitor of TMED2-DT expression is an ASO comprising a contiguous nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or preferably 100%, complementary to a TMED2-DT transcript and of length of nucleotide sequence ranging from 10 to 50 nucleotides, preferably ranging from 10 to 40, 10 to 39, 10 to 38, 10 to 37, 10 to 36, 10 to 35, 10 to 34, 10 to 33, 10 to 32, 10 to 31 or from 10 to 30 nucleotides, more preferably ranging from 11 to 29, 12, to 28, 13 to 27, 14 to 26 or from 15 to 25 nucleotides.

In one embodiment, the inhibitor of TMED2-DT expression is an ASO comprising a 2’- O-Me, 2’-F, MOE, LNA and/or cEt -modified nucleotide.

In one embodiment, the inhibitor of TMED2-DT expression is a gapmer.

The invention further elates to a pharmaceutical composition comprising the inhibitor of TMED2-DT expression according to the invention, and a pharmaceutically acceptable excipient.

The invention also relates to the inhibitor of TMED2-DT expression according to the invention, for use as a medicament. The invention also relates to the inhibitor of TMED2-DT expression according to the invention, for use in the treatment of cancer in a subject.

In one embodiment the cancer is melanoma.

In one embodiment, the inhibitor of TMED2-DT expression for use according to the invention is for use in combination with an immune checkpoint inhibitor.

In one embodiment, the cancer is, or is at risk of being, a cancer resistant to an immune checkpoint inhibitor and/or to a therapeutic agent for targeted therapy.

DEFINITIONS

Unless indicated otherwise, the following terms have the following meaning.

The singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a compound” means one compound or more than one compound.

“Antisense oligonucleotide” or “ASO” are used herein interchangeably in reference to a synthetic single stranded antisense oligonucleotide the sequence of which is at least partially complementary to the RNA sequence(s) of a target gene, and that upon binding to said RNA sequence(s), inhibits the expression of said gene. The term includes, without being limited to, “translation-blocking ASO” that impair translation of the RNA sequence(s), ASO interfering with splicing, 5’ cap formation, polyadenylation and/or ASO inducing the degradation of the targeted RNA sequence(s), in particular RNAse H- mediated degradation of the targeted RNA sequence(s). RNAse H is a cellular enzyme which recognizes duplex between DNA and RNA, and enzymatically cleaves the RNA molecules. Accordingly, to induce RNAse H mediated degradation, the ASO interfering specifically with the expression of the target gene comprises a region that comprises DNA or DNA-like nucleotides complementary to the targeted RNA which is responsible for RNAse H recruitment, ultimately leading to the cleavage of the targeted RNA sequence(s). “Complementary” when used in reference to a nucleic acid sequence, means capable of hybridizing specifically with another given (target) sequence. It is thus not absolutely required that all the bases in the region of complementarity are capable of pairing with bases in the opposing strand. Mismatches may be tolerated to some extent, as long as in the circumstances, a stretch of nucleotides is capable of hybridizing to its complementary part in the target. “Hybridizing” means the pairing or annealing to a target, herein under physiological conditions, typically via hydrogen bonding between complementary nucleotides, such as Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding. Hybridizing specifically means that the pairing or annealing is specific to the target (no off-targets effects, or at least no substantial off-targets effects).

“Expression” when used herein in reference to a gene, refers to transcription and/or translation of said gene.

“Gapmer” is used herein in reference to an ASO comprising an internal segment having a plurality of nucleotides that support RNase H cleavage, positioned between external segments, each having one or more nucleotides, wherein the nucleotide comprised in the internal segment are chemically distinct from the immediately adjacent nucleotide(s) comprised in the external segments. The internal or central segment may be referred to as the “gap”, “gap segment” or “gap region” (G); while the external segments may be referred to as the “wings”, “flanks”, “wing segments”, “flank segments”, “wing regions” or “flank regions” (F for the 5’ flank region and F’ for the 3’ flank region). Typically, the F and F’ regions are composed of modified ribonucleotides which are complementary to a target nucleic acid; whereas the G region is composed of deoxyribonucleotides, i.e. DNA or DNA-like molecules, which is responsible for RNAse H recruitment which ultimately leads to the degradation of the target nucleic acid. A gapmer is therefore a chimeric ASO. Gapmers can typically comprise a gap region (G) of 5 to 15 deoxynucleotides flanked by wing regions (F and F’) of 2 to 10 modified nucleotides each.

“Pharmaceutically acceptable excipient” is used herein in reference to an inactive or inert, and therefore nontoxic, component, as it has no pharmacological action itself, which can be used to improve properties of a composition, such as shelf-life, retention time at the application site, consumer acceptance, etc. It includes, without being limited to, surfactants (cationic, anionic, or neutral); surface stabilizers; other enhancers, such as preservatives, wetting or emulsifying agents; solvents; buffers; salt solutions; dispersion medium; isotonic and absorption delaying agents, and the like; that are physiologically compatible.

“Ribozyme” is used herein in reference to catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single- stranded nucleic acid, such as an mRNA or long non-coding RNA to which they have a complementary region. Ribozyme molecules specific for a target (herein TMED2-DT) can be designed, produced, and administered by methods commonly known to the art (see e.g., Fanning and Symonds (2006) RNA Towards Medicine (Handbook of Experimental Pharmacology), ed. Springer p. 289-303).

“RNAi agent” is used herein in reference to a nucleic acid that can inhibit expression of a target gene by RNA interference (RNAi) mechanism. RNAi agents are well-known in the art, and include, without being limited to, short-hairpin RNA (shRNA), small interfering RNA (siRNA), and double- stranded RNA (dsRNA) and nucleic acid coding for the double- stranded RNA (dsRNA) or short-hairpin RNA (shRNA). RNA interference designates a phenomenon that specifically suppresses expression of a target gene at post- transcriptional level by short interfering RNA called siRNA; the latter will bind to another enzyme (RISC) that will catalyze the cleavage of both the siRNA and target mRNA. RNAi can be initiated by double- stranded RNA molecules (dsRNA or shRNA) that, when introduced into a cell are cleaved by Dicer into a mixture of double stranded siRNA. In mammalian cells, the siRNAs that are naturally produced by Dicer are typically 21-23 bp in length, with a 19 or 20 nucleotides duplex sequence, two-nucleotide 3’ overhangs and 5 ’-triphosphate extremities.

A “therapeutically effective amount” means herein an amount that is sufficient to achieve the effect for which it is indicated, herein the treatment of cancer. The amount of the inhibitor of TMED2-DT expression of the invention to be administered can be determined by standard procedures well known by those of ordinary skill in the art. Physiological data of the patient (e.g. age, size, and weight), the routes of administration and/or the disease to be treated may have to be taken into account to determine the appropriate dosage. The amount may also vary according to other aspect of a treatment protocol (e.g. administration of other medicaments and the like).

DETAILED DESCRIPTION

Preferred characteristics and embodiments of the compounds, processes, methods, compositions, and uses of this invention are set herein below. Each characteristic and embodiment of the invention so defined may be combined with any other characteristic and/or embodiment, unless clearly indicated to the contrary. In particular, any feature or embodiment indicated as being preferred or advantageous may be combined with any other characteristic or embodiments indicated as being preferred or advantageous.

The present invention relates to an inhibitor of TMED2-DT expression.

Unless specified otherwise, the term “TMED2-DT” is used herein in reference to the gene TMED2 Divergent Transcript of NCBI Entrez genelD reference 101927415, of Ensembl reference ENSG00000247373 (in GRCh38.pl4), and/or of HUGO Gene Nomenclature Committee reference HGNC: 53346 and encompasses all transcripts, isoforms, or splice variants thereof. Database reference are made herein to entries their current release as of September 26, 2023.

In one embodiment, TMED2-DT is the transcript, isoform, or splice variant, of Ensembl reference ENST00000498967.3 (in GRCh38.pl4). ENST00000498967.3 is the sole annotated transcript of the TMED2-DT gene as of September 26, 2023. it has the nucleotide sequence SEQ ID NO. 12.

“inhibitor of TMED2-DT expression” refers to a molecule or technical means to decrease or abolish the expression of TMED2-DT. In the context of the present invention, it shall be understood that the inhibitor is selective, or in other words, specific for TMED2-DT, in that it does not inhibit, or at least does not substantially inhibit, expression of any other target. In one embodiment, the inhibitor of TMED2-DT expression is selected from the group consisting of antisense oligonucleotide (ASO), RNAi agent and ribozyme; preferably the inhibitor of TMED2-DT expression is an ASO or an RNAi agent.

It is to be understood in the context of the invention that the antisense oligonucleotide (ASO), RNAi agent or ribozyme inhibiting TMED2-DT expression, comprise a sequence, hybridizing specifically with, complementary to, or binding specifically to, a TMED2- DT transcript. The selective or specific inhibition of the expression of the gene TMED2- DT is achieved by means of the inclusion of a sequence complementary to (hybridizing specifically with) a TMED2-transcript in the ASO, RNAi agent or Ribozyme of the invention.

In other words, the antisense oligonucleotide (ASO), RNAi agent and/or ribozyme of the invention (specifically and/or directly) targets TMED2-DT, preferably (specifically and/or directly) targets a TMED2-DT transcript.

In one embodiment, the ASO of the invention induces degradation of TMED2-DT transcript, preferably induces RNAse H-mediated degradation of TMED2-DT transcript.

The inventors have found that, albeit annotated as a long noncoding RNA (IncRNA), a protein of sequence SED ID NO. 1 is translated from the open Reading Frame (ORF) of SEQ ID NO. 2 found in the third exon of ENST00000498967.3.

SEQ ID NO. 1

VAGTTGAYHHAWLIFCIFSRDVGSLCWPGWSRTPDLRRSTHLSLPKCWNYRHE PLHLAKCAFLRLSPQIDWHELMCHQVHESSNRTNSWHMVGTPYISDG

SEQ ID NO. 2

GTAGCTGGGACTACAGGCGCCTACCACCATGCCTGGCTAATTTTTTGTATTT TTAGTAGAGACGTGGGTTCACTATGTTGGCCAGGCTGGTCTCGAACTCCTGA CCTCAGGCGATCCACCCACCTCAGCCTCCCAAAGTGCTGGAATTACAGGCA TGAGCCACTGCACCTGGCCAAATGTGCATTTTTAAGGTTATCACCACAGATT GATTGGCATGAGCTCATGTGCCATCAGGTGCATGAATCATCCAACAGGACA AACAGCTGGCACATGGTGGGTACTCCATACATATCTGATGGATAA

In one embodiment, the ASO of the invention is a translation-blocking ASO, preferably a translation-blocking ASO preventing translation of the protein of amino-acid SEQ ID NO. 1.

In one embodiment, the ASO of the invention has an overall nucleotide sequence length of at least 10 nucleotides, preferably of at least 11, 12, 13, 14, 15, 16, 17 or 18 nucleotides, more preferably of at least 19 or 20 nucleotides

In one embodiment, the ASO of the invention has an overall nucleotide sequence length inferior or equal to 50 nucleotides, preferably inferior or equal to 40, 39, 38, 37, 36, 35, 34, 33, 32, or 31 nucleotides, more preferably inferior or equal to 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 or 20 nucleotides.

In one embodiment, the ASO of the invention has an overall nucleotide sequence length ranging from 10 to 50 nucleotides, preferably ranging from 10 to 40, 10 to 39, 10 to 38, 10 to 37, 10 to 36, 10 to 35, 10 to 34, 10 to 33, 10 to 32, 10 to 31 or 10 to 30 nucleotides, more preferably ranging from 11 to 29, 12, to 28, 13 to 27, 14 to 26 or 15 to 25 nucleotides.

In one embodiment, the ASO of the invention has an overall nucleotide sequence length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.

The ASO of the invention comprises a nucleotide sequence, preferably a contiguous nucleotide sequence, that is complementary to a TMED2-DT transcript.

In one embodiment, the ASO of the invention comprises a nucleotide sequence, preferably a contiguous nucleotide sequence complementary to a TMED2-DT transcript that is at least 10 nucleotides in length of nucleotide sequence, preferably at least 11, 12, 13, 14, 15, 16, 17 or 18 nucleotides in length of nucleotide sequence, more preferably of at least 19, 20 or 21 nucleotides in length of nucleotide sequence. In one embodiment, the ASO of the invention comprises a nucleotide sequence, preferably a contiguous nucleotide sequence complementary to a TMED2-DT transcript that is at most 50 nucleotides in length of nucleotide sequence, preferably at most 40, 39, 38, 37, 36, 35, 34, 33, 32, or 31 nucleotides in length of nucleotide sequence, more preferably at most 30, 29, 28, 27, 26, 25, 24, 23, 22 or 21 nucleotides in length of nucleotide sequence.

In one embodiment, the ASO of the invention comprises a nucleotide sequence, preferably a contiguous nucleotide sequence complementary to a TMED2-DT transcript of length of nucleotide sequence ranging from 10 to 50 nucleotides, preferably ranging from 10 to 40, 10 to 39, 10 to 38, 10 to 37, 10 to 36, 10 to 35, 10 to 34, 10 to 33, 10 to 31 or 10 to 30 nucleotides, more preferably of length of nucleotide sequence ranging from 11 to 29, 12, to 28, 13 to 27, 14 to 26 or 15 to 25 nucleotides.

While preferred, perfect complementarity is not necessary. In one embodiment, the contiguous nucleotide sequence complementary to a TMED2-DT transcript is complementary to a TMED2-DT transcript over its entire length. In one embodiment, the contiguous nucleotide sequence complementary to a TMED2-DT transcript comprises 1, 2, 3, 4, 5 or more mismatches.

In one embodiment, the ASO of the invention comprises, or consists of, a contiguous nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or preferably 100%, complementary to a TMED2-DT transcript and of length of nucleotide sequence ranging from 10 to 50 nucleotides, preferably ranging from 10 to 40, 10 to 39, 10 to 38, 10 to 37, 10 to 36, 10 to 35, 10 to 34, 10 to 33, 10 to 31 or 10 to 30 nucleotides, more preferably of length ranging from 11 to 29, 12, to 28, 13 to 27, 14 to 26 or 15 to 25 nucleotides.

In one embodiment, the ASO of the invention comprises (modified) ribonucleotides and/or (modified) deoxyribonucleotides. In embodiments wherein the ASO of the invention induces RNAse H-mediated degradation of a DMED2-DT transcript, the ASO of the invention comprises a region comprising (modified) deoxyribonucleotides that is complementary to said DMED2-DT transcript. In one embodiment, the ASO of the invention comprises modifications aiming at improving stability, potency and/or selectivity of said ASO.

In one embodiment, the ASO of the invention comprises phosphothioate bonds in place of phosphodiester bonds. In one embodiment, all phosphodiester bonds within the ASO of the invention are replaced by phosphothioate bonds.

In one embodiment, the ASO of the invention comprises a 2’-0-Me, 2’-F, MOE, LNA and/or cEt -modified nucleotide. In one embodiment, the ASO of the invention comprises LNA -modified nucleotides.

“2’-0-Me” refers to a nucleotide modification consisting of the substitution on position 2’ of the ribose of a 2’-O-methyl group. “2’-F” refers to a nucleotide modification consisting of the substitution on position 2’ of the ribose of a 2’ -fluoro group. “MOE” refers to a nucleotide modification consisting of the substitution on position 2’ of the ribose of a 2’-O-methoxyethyl group. “LNA” refers to Locked Nucleic Acid, a nucleotide modification consisting of the introduction of a methylene bridge between the 2’ oxygen and 4’ carbon of the ribose. “cEt” refers to Constrained Ethyl, a nucleotide modification consisting of the introduction of an ethylene bridge between the 2’ oxygen and 4’ carbon of the ribose.

In one embodiment, the ASO of the invention comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 LNA-modified nucleotides. In one embodiment, the 5’ end of the ASO of the invention consists of 1, 2, 3, 4, 5, 6, 7, 9 or 10 LNA modified nucleotides and/or the 3’ end of the ASO of the invention consists of 1, 2, 3, 4, 5, 6, 7, 9 or 10 LNA-modified nucleotides.

In one embodiment, the inhibitor of TMED2-DT expression of the invention is a gapmer.

In one embodiment, the ASO of the invention comprises, or consists of, a nucleic acid of sequence SEQ ID NO. 3. In one embodiment, the ASO of the invention comprises, or consists of, SEQ ID NO. 4.

SEQ ID NO. 3

GGTAA GGGCTAAGGG GCATG SEQ ID NO. 4 corresponds to an ASO of nucleic acid sequence SEQ ID NO. 3 wherein 5 nucleotides at the 5’ end and 5 nucleotides at the 3’ end (in bold font above) are locked nucleic acids (LNA) and wherein the central part (italicized above) is DNA and wherein the phosphodiester bonds are replaced by phosphothioate linkage in the entire sequence.

In one embodiment, the ASO of the invention comprises, or consists of, a nucleic acid of sequence SEQ ID NO. 5. In one embodiment, the ASO of the invention comprises, or consists of, SEQ ID NO. 6.

SEQ ID NO. 5

CAGCTACTCAGGAGGC

SEQ ID NO. 6 corresponds to an ASO of nucleic acid sequence SEQ ID NO. 5 wherein all nucleotides are locked nucleic acids (LNA) and wherein the phosphodiester bonds are replaced by phosphothioate linkage in the entire sequence.

The RNAi agent of the invention comprise a sequence complementary to at least one part of a TMED2-DT transcript, in particular to at least one exon thereof.

In one embodiment, the RNAi agent of the invention is selected from the group consisting of short-hairpin RNA (shRNA), small interfering RNA (siRNA), and double-stranded RNA (dsRNA) and nucleic acid coding for a double-stranded RNA (dsRNA) or shorthairpin RNA (shRNA).

In one embodiment, the RNAi agent of the invention comprises, or consists of, a contiguous nucleotide sequence of at least 10 to 40 (preferably 15 to 30, 16 to 25, 17 to 24, 20, 21, 22 or 23) nucleotides in length that is complementary to a specific region of a TMED2-DT transcript.

In one embodiment, the ribozyme of the invention, is targeting a TMED2-DT transcript; In other word the ribozyme of the invention induces cleavage of a TMED2-DT transcript.

The present invention further relates to the use of an inhibitor of TMED2-DT expression according to the invention as a medicament. The present invention also relates to a pharmaceutical composition comprising the inhibitor of TMED2-DT expression according to the invention, and a pharmaceutically acceptable excipient.

The present invention also relates to the use of an inhibitor of TMED2-DT expression according to the invention for the treatment of cancer.

In one embodiment, said cancer is selected from the group consisting of melanoma (including without being limited to skin melanoma, skin cutaneous melanoma and uveal melanoma), skin cancer (including, without being limited to skin melanoma, squamous cell carcinomas and Merkel cell carcinoma), bladder cancer (including without being limited to, bladder urothelial carcinoma), brain cancer (including without being limited to, brain lower grade glioma, glioblastoma and glioblastoma multiforme), breast cancer (including without being limited to, breast invasive carcinoma, triple negative breast cancer), adrenal gland cancer (including without being limited to, adrenocortical cancer), bile duct cancer (including without being limited to, cholangiocarcinoma), cervical cancer (including without being limited to, cervical & endocervical cancer), colon cancer (including without being limited to, colon adenocarcinoma), esophageal cancer (including without being limited to, esophageal carcinoma), head and neck cancer (including without being limited to, head & neck squamous cell carcinoma), kidney cancer (including without being limited to, chromophobe renal cell carcinoma, kidney clear cell carcinoma, kidney papillary cell carcinoma), Leukemia, liver cancer (including without being limited to, liver hepatocellular carcinoma), lung cancer (including without being limited to, lung adenocarcinoma and lung squamous cell carcinoma), lymphoma (including without being limited to, diffuse large B-cell lymphoma), melanoma (including without being limited to, melanoma and uveal melanoma), mesothelioma, ovarian cancer (including without being limited to, ovarian serous cystadenocarcinoma), pancreatic cancer (including without being limited to, pancreatic adenocarcinoma), paraganglioma, prostate cancer (including without being limited to, prostate adenocarcinoma), rectum cancer (including without being limited to, rectum adenocarcinoma), sarcoma cancer (including without being limited to, sarcoma), stomach cancer (including without being limited to, stomach adenocarcinoma), testicular cancer (including without being limited to, testicular germ cell tumor), thymoma (including without being limited to, thymoma), thyroid cancer (including without being limited to, thyroid carcinoma), uterine cancer (including without being limited to, uterine carcinosarcoma and uterine corpus endometrioid carcinoma ). In one embodiment, said cancer is selected from the group consisting of melanoma (including without being limited to skin melanoma, skin cutaneous melanoma and uveal melanoma), skin cancer (including, without being limited to skin melanoma, squamous cell carcinomas and Merkel cell carcinoma), bladder cancer (including without being limited to, bladder urothelial carcinoma), brain cancer (including without being limited to, brain lower grade glioma, glioblastoma and glioblastoma multiforme) and breast cancer (including without being limited to, breast invasive carcinoma, triple negative breast cancer). In one embodiment, said cancer is melanoma.

The present invention also relates to the use of an inhibitor of TMED2-DT expression according to the invention for the treatment of melanoma.

In one embodiment, the inhibitor of TMED2-DT expression for use in the treatment of cancer according to the invention is for use in combination with an immune checkpoint inhibitor and/or for use in combination with a therapeutic agent for targeted therapy.

Examples of immune checkpoint inhibitor that may be used in combination with the inhibitor of TMED2-DT expression of the invention include, without being limited to, anti PD-1 antibodies, such as pembrolizumab, pidilizumab and nivolumab, anti PD-L1 antibodies, such as atezolizumab, avelumab, durvalumab, anti CTLA4 antibodies such as ipilimumab and tremelimumab and anti LAG3 antibodies, such as relatlimab.

In one embodiment, the immune checkpoint inhibitor is selected from the group consisting of PD-1 inhibitors, CTLA-4 inhibitors, PD-L1 inhibitors and LAG3 inhibitors.

In one embodiment, the immune checkpoint inhibitor is selected from the group consisting of anti PD-1 antibody, anti CTLA-4 antibody, anti PD-L1 antibody, and anti LAG3 antibodies. Examples of agent for targeted therapy that may be used in combination with the inhibitor of TMED2-DT expression of the invention include, without being limited to, B-Raf inhibitors, such as dabrafenib, and MEK inhibitors, such as trametinib.

In one embodiment, the therapeutic agent for targeted therapy is selected from the group consisting of B-Raf inhibitors and MEK inhibitors.

In one embodiment, the therapeutic agent for targeted therapy is selected from the group consisting of dabrafenib and trametinib.

In one embodiment, melanoma is invasive and/or metastatic melanoma.

“Invasive melanoma” refers herein to a melanoma that is not confined to the upper layer of the epidermis.

“Metastatic melanoma” refers herein to melanoma that has metastasized.

In one embodiment melanoma is, or is at risk of being, melanoma resistant to (immuno- and/or targeted) therapy, “melanoma resistant to therapy” refers to herein to melanoma that does not, or partially, respond to said therapy.

Methods to determine the risk of a melanoma being resistant to therapy are known to the skilled artisan and include, without being limited to, the use of biomarkers, such as mutations or expression of specific gene products, indicative of a risk of therapy resistance.

In one embodiment, melanoma is, or is at risk of being, melanoma resistant to immune checkpoint inhibitor and/or to a therapeutic agent for targeted therapy.

In the context of the invention, the subject is human.

In one embodiment, the subject has, or is at risk of having, an invasive melanoma.

In one embodiment, the subject has, or is at risk of having, a metastatic melanoma.

In one embodiment, the subject has, or is at risk of having, a melanoma resistant to therapy. In one embodiment, the subject has, or is at risk of having, a melanoma resistant to immune checkpoint inhibitor and/or to a therapeutic agent for targeted therapy.

The invention further relates to a method for treating cancer in a subject in need thereof, comprising the administration to said subject of a therapeutically effective amount of an inhibitor of TMED2-DT expression according to the invention. Embodiments described above in respect to the inhibitor of TMED2-DT expression according to the invention and its uses apply herein mutatis mutandis.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing normalized expression of TMED2-DT determined by qPCR in polysome fractions from SK-MEL-28 cells upon induction of the ISR and in untreated control (Ctrl).

Figure 2 is a graph showing normalized expression of TMED2-DT determined by qPCR in polysome fractions from Mel-015 tumor samples relapsing after targeted therapy (dabrafenib and trametinib - DT) and untreated control (Vehicle).

Figure 3. Panel A: expression of TMED2-DT protein in SK-MEL-28 cells upon induction of the ISR and in vehicle-treated controls (Ctrl) as detected by Western Blotting. Panel B: expression of TMED2-DT protein in Mel-015 tumor sample relapsing after treatment with targeted therapy with dabrafenib and trametinib (DT) and in vehicle- treated control (Vehicle - Veh) as detected by Western Blotting.

Figure 4. Panel A: Overall survival of melanoma patients in the Genomic Data Commons - TCGA cohort stratified on TMED2-DT DNA copy number (Threshold value 0.004100). p: p- value in a long-rank (Mantel Cox) test. Panel B: Overall survival of cancer patients in the Genomic Data Commons - TCGA pan-cancer cohort [5] stratified on TMED2-DT DNA copy number (Threshold value 0.01310). p: -valuc in a long-rank (Mantel Cox) test. Figure 5. correlation of TMED2-DT expression and different immune signature [1] in the TCGA melanoma cohort.

Figure 6. TMED2-DT protein expression after response to anti-PD-1 in tumor samples from MEL-006 PDX melanoma model treated with Nivolumab (a-PD-1) and vehicle- treated control (Vehicle). **: -valuc in two-sided unpaired t test = 0.0022

Figure 7. efficiency of knock down of TMED2-DT Gapmer. Panel A: RT-qPCR quantification of TMED2-DT expression using TMED2-DT Fwd. and TMED2-DT Rev. primers (TMED-DT) or TMED2-DT ORF Fwd. and TMED2-DT ORF Rev. primers (ORF) in cells treated with TMED2-DT Gapmer, cells treated with a control ASO and in untreated cells (Mock). ***: -valuc in two-tailed paired t test < 0.005. . Panel B shows TMED2-DT protein expression in cells treated with TMED2-DT Gapmer (TMED2-DT KD), cells treated with a control ASO (Ctrl) and in untreated cells (Mock). Beta actin is used as a loading control.

Figure 8. quantification of a co-culturing experiment between melanoma cells and HLA- matched peripheral blood mononuclear cells (PBMCs) upon treatment with a Gapmer against TMED2-DT or with a control ASO. Panel A shows MM099 cells confluency. Panel B shows average caspase counts per image. **** p value (in both instances) calculated by two-ways ANOVA for the interaction between time and treatment was below 0.0001.

EXAMPLES

The present invention is further illustrated by the following examples.

Materials

SK-MEL-28 cells were obtained from the American Type Culture Collection. MM099 cells were obtained from prof. G. Ghanem at Institut Jules Bordet (Belgium).

The cutaneous melanoma PDX model MEL-015 and MEL-006 [3] is part of the Trace collection (https://gbiomed.kuleuven.be/english/research/50488876/54502087/Trace) and was established using metastatic melanoma lesions derived from patients undergoing surgery as part of standard treatment at UZ Leuven.

An antisense oligonucleotide was used to induce RNAse H-dependent degradation of TMED2-DT. The ASO is a Gapmer of nucleic acid sequence 5’- GGTAA GGGCTAAGG GGCATG - 3’ (SEQ ID NO. 3) wherein 5 nucleotides at the 5’ end and 5 nucleotides at the 3’ end (in bold font above) are locked nucleic acids (LNA) and wherein the central part (italicized above) is DNA and wherein the phosphodiester bonds are replaced by phosphothioate linkage in the entire sequence. The modified ASO sequence is SEQ ID NO. 4.

Methods

Polysome profiling

Tumor pieces from the Mel-015 PDX model and SK-MEL-28 cells (15-cm dishes per each condition) were plated to have 70% confluency after 72 h. The following day to induce ISR, cells were treated with 20 pM salubrinal (Sigma-Aldrich, US). 72 h after the start of the treatment, cells were treated with 100 pg/ml of cycloheximide (Sigma- Aldrich, US) for 12 min at 37°C, collected, and resuspended in lysis buffer (30 mM Tris- HC1, 150 mM KC1, 10 mM MgCh supplemented with 1 mM dithiothreitol (DTT - Sigma- Aldrich, US), 100 pg/ml cycloheximide, 20 U/pl SUPERase-IN RNase Inhibitor (Thermo Fisher Scientific, US) and Halt Protease and Phosphatase Inhibitor Single-Use Cocktail (cat# 78442 Life Technologies, US) before the start of the experiment). Lysates were then incubated agitating at 4°C for 35 min, and then centrifuged at 17 000 ref for 15 min at 4°C. Lysates were loaded on a sucrose gradient (the linear sucrose gradient 5-20% was generated from two different solutions, sucrose 5 and 20%, made with buffer G (20 mM Tris- HC1, 100 mM KC1, 10 mM MgCh supplemented with 1 mM DTT and 100 pg/ml cycloheximide before the start of the experiment). Samples were then centrifugated in an SW41Ti rotor (Beckman Coulter, US) at 37,000 rpm for 170 min at 4°C. The fractions were obtained with a BioLogic LP System (Bio-Rad, US). 14 fractions were collected from each sample, with each fraction having a final volume of 600 pl. From the initial 14 fractions, 4 final samples were obtained (by pulling together some of them): 40S, 60S, 80S, and polysomes. qPCR

RNA was reverse transcribed using the High-Capacity complementary DNA Reverse Transcription Kit (Thermo Fisher Scientific) on a Veriti 96- well thermal cycler (Thermo Fisher Scientific). Gene expression was measured by qPCR on a QuantStudio 5 (Thermo Fisher Scientific) and normalized using 28S and 18S as reference genes (for polysome profiling experiments) or the average of HPRT, TBP, and UBC. Primers with sequences indicated in Table 1 were used for TMED2-DT and for the Open Reading Frame (ORF) corresponding to TMED2-DT protein.

Table 1: primers

Colony assay

For colony formation assays, cells were plated in 6-well plates at the appropriate density and grown for 120 hours. Subsequently, cells were washed twice with PBS, fixed, and stained for 15 min with 1% crystal violet in 35% methanol solution.

Surface occupancy was measured using ImageJ.

Co-culture with PBMCs

MM099 cells were plated in a 6 well (150 000 cells per well). Knock-down (KD) of TMED2-DT was performed, 24h after seeding, with the gapmer (SEQ ID NO. 4), at the concentration of 30 nM. The same day, 1.5 million peripheral blood mononuclear cells (PBMCs) (HLA matched) were grown in 1.5 ml of RPMI, complemented with complemented with CD28 (5 ug/ul), CD3 (3 ug/ul) and IL2 (200 ng in total). 48h after transfection, the MM099 were reseeded in a 96 well plate (1500 cell per well) in fresh medium and co-cultured with PBMCs (7500 cell per well) labelled with Nuclight Rapid Red Dye. PBMCs were also cultured in the medium derived from transfected cells at 48h from transfection in the absence of melanoma cells. The ratio melanoma cells vs PBMCs was 1:5. Differences in cell numbers were calculated with incucyte over 48h.

Immuno-fluorescence staining

Cells cultured on slides were fixed on PFA 4% for 10 min. After a couple of washes in PBS the cells were permeabilized on ice for 10 minutes in 1 % BSA, 0.2% Triton X-100 in PBS. To block non-specific interactions, cells were blocked for 30 min in goat serum prior of incubation with primary antibody (2pg /ml) for 1 hour at RT. After 3 washes in permeabilization solution the cells were incubated with anti-rabbit alexa fluor conjugated for 45 min before mounting them in DAPI containing medium.

Protein extraction and Western blot

Proteins were extracted resuspending cellular pellet in RIPA buffer [150 mM NaCl, 50 mM Tris-HCl pH 8, 1% Nonidet P40 (Thermo Fisher Scientific), 0.5% Sodium Deoxycholate (Sigma Aldrich), ImM EDTA (Sigma Aldrich)] supplemented with lx Halt Protease and Phosphatase Inhibitor Single-Use Cocktail (Life Technologies). 50ug of samples were loaded on 10-20% Tricine 1.0mm miniprotein gels and transferred on a nitrocellulose membrane for detection with a rabbit TMED2-DT antibody or anti-actin (cell signalling, Rabbit 1:3000).

MS-based proteomics analysis

Both control and salubrinal-treated cell pellets were digested using the PreOmics iST sample preparation kit, following the manufacturer’s guidelines. In all cases, proteolytic peptides were separated by re versed-phase chromatography on an EASY-nLC 1200 ultra- high-performance liquid chromatography (UHPLC) system through an EASY-Spray column (Thermo Fisher Scientific), 25-cm long (inner diameter 75 pm, PepMap C18, 2 pm particles), which was connected online to a Q Exactive HF (Thermo Fisher Scientific) instrument through an EASY-Spray™ Ion Source (Thermo Fisher Scientific). Both for library and study samples, the purified peptides were loaded in buffer A (0.1% formic acid in water) at constant pressure of 980 Bar. They were separated through the following gradient: 30 min of 3-23% of buffer B (0.1% formic acid, 80% acetonitrile), 5 min 23- 30% of buffer B, 1 min 60-95% buffer B, at a constant flow rate of 250 nl/min. The column temperature was kept at 45°C under EASY-Spray oven control. The mass spectrometer was operated data-dependent acquisition (DDA) mode. Briefly, MS spectra were collected in the Orbitrap mass analyzer at a 60,000 resolution (200 m/z) within a range of 300-1550 m/z with an automatic gain control (AGC) target of 3e6 and a maximum ion injection time of 20 ms. The 15 most intense ions from the full scan were sequentially fragmented with an isolation width of 1.4 m/z, following higher-energy collisional dissociation (HCD) with a normalized collision energy (NCE) of 28%. The resolution used for MS/MS spectra collection in the Orbitrap was 15,000 at 200 m/z with an AGC target of le5 and a maximum ion injection time of 80 ms. Precursor dynamic exclusion was enabled with a duration value of 20s. MS Raw files were processed with MaxQuant (MQ) version 2.0.3.0 integrated with Andromeda search engine. Sequenced non-coding RNAs were in silico translated into amino acid sequences already in FASTA format, using the “Six-frame translation” tool available in the MQ suite. In parallel, MSMS spectra were also searched against the human reference proteomes (Uniprot UP000005640, 80,027 entries). The search included cysteine carbamidomethylation as a fixed modification and methionine oxidation and acetylation of the protein N-terminus as variable modifications. Required minimum peptide length was 7 amino acids and maximum mass tolerances were 4.5 p.p.m. for precursor ions after nonlinear recalibration and 20 p.p.m. for fragment ions. Identifications were stringently filtered for a FDR < 1% at both peptide spectrum match and protein group levels.

Results and conclusions

Translational rewiring via the activation of the Integrated Stress Response (ISR) is a major evolutionary-conserved driver of invasiveness and therapy resistance [2,3]. The ISR consists in a global reduction of CAP-dependent translation, replaced by an IRES- dependent mechanism [2]. We identified an annotated IncRNA, TMED2-DT, normally enriched at polysome, shifting back to the 40S upon induction of the ISR (figure 1). Similar results were obtained upon chronic activation of the ISR with prolonged treatment with targeted therapy (dabrafenib and trametinib) in a BRAF mutant melanoma PDX model (figure 2). When performing prediction of coding potential for the 473 IncRNA differentially associated with the ribosome upon induction of the ISR, as described in [4], we found that TMED2-DT has a coding probability of 0.75, in line with its enrichment at polysomes in untreated cells. We have therefore performed proteomics to search for peptides potentially derived from the translation of TMED2-DT in all three reading frames. A peptide (SEQ ID NO. 11 indicated in bold font within the protein sequence SEQ ID NO. 1 above) derived from a protein of the predicted molecular weight of

~12KDa and of sequence SEQ ID NO. 1 (herein “TMED2-DT protein”), encoded by the ORF of sequence SEQ ID NO. 2, was identified.

Downregulation of TMED2-DT protein after ISR induction and in melanoma PDX that have acquired resistance to targeted therapy was confirmed by western blot (figure 3A and B). Copy number of TMED2-DT was found be altered in several cancers (Table 2) and to significantly correlates with poor patient survival in the TCGA melanoma and pancancer cohorts (figure 4 and B).

Table 2: copy number of TMED2-DT in TCGA cohorts. CN: copy number; QI: first quartile; Q3: third quartile.

TMED2-DT was furthermore found be expressed in several cancer (Table 3) and its expression was found significantly lower in inflamed melanoma in the TCGA cohort [1] (figure 5). Table 3: Expression of TMED2-DT in several cancers in TCGA cohorts, fpkm: Fragments Per Kilobase of transcript per Million mapped reads ; QI: first quartile; Q3: third quartile.

In keeping with this, expression of the TMED2-DT protein decreases in response to immune checkpoint blockade in patient-derived xenograft models responding to immune checkpoint blockade (figure 6). We designed an AntiSense Oligonucleotide for the RNAse H-dependent knock down of TMED2-DT, that was confirmed at RNA and protein level in SK-MEL-28 (figure 7A and B). Inhibition of TMED2-DT has only minor effects on melanoma viability, however when coculturing melanoma cells with HLA-matched PBMCs we detected increased immune-mediated melanoma killing (figure 8A and B). Following TMED2-DT inhibition, the PBMCs show increased degranulation this is indicative of an increase in effector T-cell functions.

REFERENCES

1- Thorsson V, Gibbs DL, Brown SD, et al. The Immune Landscape of Cancer [published correction appears in Immunity. 2019 Aug 20;51(2):411- 412], Immunity. 2018;48(4):812-830.el4. doi:10.1016/j.immuni.2018.03.023.

2- Licari, E., L. Sanchez-del-Campo, and P. Falletta, The two faces of the Integrated Stress Response in cancer progression and therapeutic strategies. The International Journal of Biochemistry & Cell Biology, 2021. 139: p. 106059.

3- Vendramin, R., et al., Activation of the integrated stress response confers vulnerability to mitoribosome-targeting antibiotics in melanoma. J Exp Med, 2021. 218(9)7

4- Cinque S, Verheyden Y, Katopodi Z, Knezevic Z, Demesmaeker E, Adnane S, Hanache S, Vendramin R, Stinkens F, Vervloesem F, Cuomo A, Pozniak J, Cortes Calabuig A, Tabruyn S, Bechter O, Baietti MF, Groaz E, Bonaldi T, Eeucci E. The cancer-specific IncRNA LISR customizes ribosomes to suppress anti-tumour immunity bioRxiv 2023.01.06.523012; doi: https://doi.org/10.1101/2023.01.06.523012.

5- Zhang, Z., et al., Uniform genomic data analysis in the NCI Genomic Data Commons. Nature Communications, 2021. 12(1): p. 1226

Claims

1. A specific inhibitor of TMDE2-DT expression selected from the group consisting of antisense oligonucleotide (ASO), RNAi agent and ribozyme, for use in the treatment of cancer, wherein said ASO, RNAi agent or ribozyme comprises a nucleotide sequence complementary to a TMED2-DT transcript.

2. The specific inhibitor of TMED2-DT expression for use according to claim 1, wherein said inhibitor of TMED2-DT expression is an ASO inducing degradation of a TMED2-DT transcript, preferably inducing RNAse H-mediated degradation of a TMED2-DT transcript.

3. The specific inhibitor of TMED2-DT expression for use according to claim 1, wherein said inhibitor of TMED2-DT expression is a translation-blocking ASO, preferably a translation-blocking ASO preventing translation of the protein of amino acid sequence SEQ ID NO. 1.

4. The specific inhibitor of TMED2-DT expression for use according to claim 1, wherein said RNAi agent is selected from the group consisting of siRNA, shRNA, dsRNA and nucleic acid coding for a dsRNA or for a shRNA.

5. The specific inhibitor of TMED2-DT expression for use according to any one of claims 1 to 4, wherein said inhibitor of TMED2-DT expression is an ASO having an overall nucleotide sequence length of at least 10 nucleotides, preferably of at least 11, 12, 13, 14, 15, 16, 17 or 18 nucleotides, more preferably of at least 19 or 20 nucleotides.

6. The specific inhibitor of TMED2-DT expression for use according to any one of claims 1 to 5, wherein said inhibitor of TMED2-DT expression is an ASO comprising a contiguous nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or preferably 100%, complementary to a TMED2-DT transcript and of length of nucleotide sequence ranging from 10 to 50 nucleotides, preferably ranging from 10 to 40, 10 to 39, 10 to 38, 10 to 37, 10 to 36, 10 to 35, 10 to 34, 10 to 33, 10 to 32, 10 to 31 or from 10 to 30 nucleotides, more preferably ranging from 11 to 29, 12, to 28, 13 to 27, 14 to 26 or from 15 to 25 nucleotides.

7. The specific inhibitor of TMED2-DT expression for use according to any one of claims 1 to 6, wherein said inhibitor of TMED2-DT expression is an ASO comprising a 2’-0-Me, 2’-F, MOE, LNA and/or cEt -modified nucleotide.

8. The specific inhibitor of TMED2-DT expression for use according to any one of claims 1 to 7, wherein said inhibitor of TMED2-DT expression is a gapmer.

9. The specific inhibitor of TMED2-DT expression for use according to any one of claims 1 to 8, wherein said cancer is melanoma.

10. The inhibitor of TMED2-DT expression for use according to any one of claims 1 to 9, for use in combination with an immune checkpoint inhibitor.

11. The inhibitor of TMED2-DT expression for use according to any one of claims 1 to 10, wherein said cancer is, or is at risk of being, a cancer resistant to an immune checkpoint inhibitor and/or to a therapeutic agent for targeted therapy.

12. A specific inhibitor of TMED2-DT expression selected from the group consisting of antisense oligonucleotide (ASO), RNAi agent and ribozyme and wherein said ASO, RNAi agent or ribozyme comprises a nucleotide sequence complementary to a TMED2-DT transcript.

13. The specific inhibitor of TMED2-DT expression according to claim 12, wherein said inhibitor of TMED2-DT expression is an ASO inducing degradation of a TMED2-DT transcript, preferably inducing RNAse H-mediated degradation of a TMED2-DT transcript.

14. The specific inhibitor of TMED2-DT expression according to claim 12, wherein said inhibitor of TMED2-DT expression is a translation-blocking ASO, preferably a translation-blocking ASO preventing translation of the protein of amino acid sequence SEQ ID NO. 1.

15. The specific inhibitor of TMED2-DT expression according to claim 12, wherein said RNAi agent is selected from the group consisting of siRNA, shRNA, dsRNA and nucleic acid coding for a dsRNA or for a shRNA.

16. The specific inhibitor of TMED2-DT expression according to any one of claims 12 to 15, wherein said inhibitor of TMED2-DT expression is an ASO having an overall nucleotide sequence length of at least 10 nucleotides, preferably of at least 11, 12, 13, 14, 15, 16, 17 or 18 nucleotides, more preferably of at least 19 or 20 nucleotides.

17. The specific inhibitor of TMED2-DT expression according to any one of claims 12 to 15, wherein said inhibitor of TMED2-DT expression is an ASO comprising a contiguous nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or preferably 100%, complementary to a TMED2-DT transcript and of length of nucleotide sequence ranging from 10 to 50 nucleotides, preferably ranging from 10 to 40, 10 to 39, 10 to 38, 10 to 37, 10 to 36, 10 to 35, 10 to 34, 10 to 33, 10 to 32, 10 to 31 or from 10 to 30 nucleotides, more preferably ranging from 11 to 29, 12, to 28, 13 to 27, 14 to 26 or from 15 to 25 nucleotides.

18. The specific inhibitor of TMED2-DT expression according to any one of claims 12 to 17, wherein said inhibitor of TMED2-DT expression is an ASO comprising a 2’-0-Me, 2’-F, MOE, LNA and/or cEt -modified nucleotide.

19. The specific inhibitor of TMED2-DT expression according to any one of claims 12 to 18, wherein said inhibitor of TMED2-DT expression is a gapmer.

20. A pharmaceutical composition comprising the specific inhibitor of TMED2-DT expression according to any one of claims 12 to 19, and a pharmaceutically acceptable excipient.

21. The specific inhibitor of TMED2-DT expression according to any one of claims 12 to 19, for use as a medicament.