WO2012016050A2 - Chemoradiotherapy for kras-mutant colorectal cancer - Google Patents

Abstract

This invention relates to methods of treating KRAS-mutant cancers by administering a composition comprising Midostaurin (PKC-412), and methods for increasing the sensitivity of colorectal cancers to radiation, e.g., using Midostaurin (PKC-412) to radiosensitize colorectal cancers or cancer cells.

Description

Chemoradiotherapy for KRAS-Mutant Colorectal Cancer

CLAIM OF PRIORITY

This application claims the benefit of U.S. Provisional Patent Application Serial No. 61/368,482, filed on July 28, 2010, the entire contents of which are hereby incorporated by reference.

TECHNICAL FIELD

This invention relates to methods of increasing the sensitivity of colorectal cancers to radiation, and more particularly to the use of Midostaurin (PKC-412) to radiosensitize colorectal cancers or cancer cells.

BACKGROUND

Cancers of the colon and rectum are highly similar biologically, but differ with respect to their clinical management. Whereas Stage I rectal cancer is often cured with surgical resection alone, multimodal therapy is needed for patients with locally advanced (Stage II-III) disease, where the risk of local recurrence is significant (Swedish Rectal Cancer Trial, The New England Journal of Medicine 336, 980-987 (1997); Gastrointestinal Tumor Study Group, The New England Journal of Medicine 312, 1465-1472 (1985)). The requirement for multimodal therapy has been demonstrated in randomized trials, where it was shown to decrease local disease recurrence and to improve survival. For example, the Gastrointestinal Tumor Study Group, randomizing 227 patients with Dukes' B2 and C rectal cancer (corresponding to AJCC Stage II-III) after surgery to observation, adjuvant radiation, chemotherapy (5-FU/semustine), or chemoradiation, found that adjuvant chemoradiation significantly lowered disease recurrence and improved survival (Gastrointestinal Tumor Study Group, The New England Journal of Medicine 312, 1465-1472 (1985)). Neoadjuvant chemoradiation may further improve clinical outcomes. In the NSABP trial R-03, which randomized patients to neoadjuvant or adjuvant chemoradiation using 5-FU/leucovorin, followed by 4 cycles of consolidation 5-FU/leucovorin, neoadjuvant treatment statistically improved 3-year disease-free survival (70 vs. 65%) and trended toward improvement in overall survival (85 vs. 78%), though this difference were not statistically significant. Based on this, and similar, studies, neoadjuvant chemoradiation using 5-FU-based chemotherapy for Stage II-III rectal cancer, followed by surgical resection and consolidation chemotherapy, has become the standard practice in many institutions.

SUMMARY

Colorectal cancer strikes nearly 150,000 Americans per year and nearly half of these cancers have activating mutations in the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) oncogene. These mutations are among the best predictive biomarkers for the failure of a cancer to respond to both conventional and targeted therapies, including ionizing radiation. As shown herein, the administration of Midostaurin (also called PKC-412) can dose-dependently induce apoptosis and reverse the radiation insensitivity of these cancers.

In one aspect, the invention provides methods for increasing sensitivity of a mutant KRAS associated cancer to radiation. The methods include identifying a cancer cell having a mutant KRAS gene; administering a therapeutically effective dose of Midostaurin to the cell; and administering a therapeutically effective dose of radiation to the cell, thereby increasing the sensitivity of the cell to radiation therapy.

In another aspect, the invention provides methods for treating a subject who has a mutant KRAS associated cancer. The methods include identifying a subject who has a cancer that is associated with a mutant KRAS gene; administering a

therapeutically effective dose of Midostaurin to the subject; and administering a therapeutically effective dose of radiation to the subject, thereby treating the subject.

In a further aspect, the invention provides methods of selecting a treatment for a subject who has cancer, the method comprising: providing a sample comprising a cancer cell from the subject; detecting the presence of mutant KRAS in the cancer cell; and selecting a treatment comprising administering Midostaurin and radiation for the subject if the cancer cell has a mutant KRAS. In some embodiments, the methods further include administering the treatment to the subject.

In a further aspect, the invention provides methods for treating a subject who has a mutant KRAS associated cancer. The methods include identifying a subject who has a cancer that is associated with a mutant KRAS gene; and administering a therapeutically effective dose of Midostaurin to the subject; thereby treating the subject.

In yet another aspect, the invention features methods for selecting a treatment for a subject who has cancer. The methods include providing a sample comprising a cancer cell from the subject; detecting the presence of mutant KRAS in the cancer cell; and selecting a treatment comprising administering Midostaurin for the subject if the cancer cell has a mutant KRAS. In some embodiments, the methods further include administering the treatment to the subject.

In some embodiments, the identifying step comprises obtaining a cell of the cancer and detecting the presence of a mutant KRAS in the cell.

In some embodiments, detecting the presence of a mutant KRAS includes detecting expression of a mutant K-RAS protein, or presence of a mutant KRAS nucleic acid. In some embodiments, detecting presence of a mutant KRAS nucleic acid comprises detecting presence of a KRAS transcript or gene comprising an activating mutation. In some embodiments, the activating mutation comprises an amino acid substitution of residue G12, G13, Q22, E31, Q61, K117 or A146 of the mature protein.

In some embodiments, the methods further include administering a chemotherapeutic agent, e.g., 5-flurouracil (5-FU).

In a further aspect, the invention describes the use of Midostaurin, alone or with 5-FU in the treatment of a KRAS-mutation associated cancer. In some embodiments, radiation is also administered.

In some embodiments, the cancer is selected from the group consisting of lung adenocarcinoma, mucinous adenoma, pancreatic cancer, breast cancer, bladder cancer, thyroid cancer, gastric cancer, cholangiocarcinoma, or colorectal carcinoma; in some embodiments, the cancer is rectal carcinoma.

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. For example, KRAS refers to the oncogene and K-RAS refers to the oncoprotein. 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

FIGs 1A-B show that Midostaurin affects overall viability in cells expressing mutant K-RAS by inducing apoptosis. FIG. 1A is a line graph showing that when compared to DKs-8 cells, which express only wild-type K-RAS, KRAS-mutant DLD- 1 cells are hyper-sensitive to Midostaurin, a broad-spectrum kinase inhibitor. DLD- 1 and DKs-8 are genetically-identical p53^_/~ colorectal cancer cell lines, with the exception of the KRAS activating mutation (Shirasawa et al, (1993). Science 260, 85- 88). FIG. IB is a bar graph illustrating the dose-dependent induction of apoptosis by Midostaurin. Apoptosis was quantified by FACS for Annexin V.

FIGs. 2A-B show that Midostaurin reverses the radiation resistance associated with mutant K-RAS. FIG. 2A is a line graph showing that at therapeutically-relevant doses (approximately 2 Gy), mutant K-RAS confers resistance to ionizing radiation. Pretreatment with Midostaurin (100 nM) increases response (i.e., decreases survival) in KRAS-mutant DLD-1 cells, but has no effect on KRAS wild-type DKs-8 cells. Radiation response was measured via clonogenic survival assay. FIG. 2B is a bar graph showing the results of experiments in which clonogenic survival was used to measure the effect of Midostaurin +/- chemotherapy (5-FU) in cells expressing mutant K-RAS. Chemotherapy alone had no effect, but it cooperated with Midostaurin to enhance response.

FIG. 3 provides the protein sequences for human K-RAS variants (a) and (b). The commonly mutated residues are underlined.

FIG. 4 is a bar graph showing the effects of midostaurin treatment on phospho-c-Jun levels.

DETAILED DESCRIPTION

As described herein, the presence of mutationally activated K-RAS confers resistance to ionizing radiation, a common treatment modality, in combination with chemotherapy, for advanced rectal cancer. By extension, it was hypothesized that the efficacy of neoadjuvent chemoradiation would be significantly improved by a targeted therapy that inhibits the radiation resistance phenotype associated with mutant K- RAS. Unfortunately, no targeted therapies currently exist to inhibit KRAS function. Using clonogenic survival assays to measure radiation sensitivity, a screen for small molecules that can inhibit the ability of mutant K-RAS to confer resistance to ionizing radiation was performed. As described herein, Midostaurin (PKC-412), a broad- spectrum kinase inhibitor, induces apoptosis and abrogates the radiation resistance associated with mutant K-RAS. Thus, described herein are methods for treating cancer, and for increasing tumor and tumor cell sensitivity to therapy, e.g., to radiation therapy.

K-RAS and the Clinical Significance ofKRAS Mutations

K-RAS is a proto-oncoprotein belonging to the RAS superfamily of small monomeric GTPases. It normally functions downstream of receptor tyrosine kinases and upstream of kinase-based signaling cascades, for example the

RAF→MEK→ERK and PI3K→AKT pathways. The KRAS gene produces two distinct, but highly related, 21 kD proteins through alternative splicing: (1) GTPase K- RAS isoform (b) precursor (GenBank Acc. Nos. NM_004985.3 (nucleic acid) and NP_004976.2 (protein)) and (2) GTPase K-RAS isoform (a) precursor (GenBank Acc. Nos. NM_033360.2 (nucleic acid) and NP_203524.1 (protein)). Variant (b) is composed of five exons, and terminates in exon 4b, thus lacking exon 4a, which the longer transcript, variant (a), includes (variant (a) has all six exons).

Single amino acid substitutions, e.g., at codons 12 (e.g. G12A, G12V, G12D, G12S, G12R, G12N, or G12C), 13 (e.g., G13C or G13D), 22 (e.g., Q22K), 31 (e.g., E31K), 61 (e.g., Q61L, Q61H, or Q61R), 117 (e.g., K117N), or 146 (e.g., A147T or A146V) of SEQ ID NO: 1 (since in this region variants (a) and (b) are identical, for simplicity the following will refer to SEQ ID NO: 1, which is variant (b), but as one of skill in the art will appreciate a mutation in the gene would produce mutated versions of both variants (a) and (b)), are responsible for oncogenic activity. See, e.g., Janakiraman et al. Cancer Research 70: 5901-5911 (2010). The transforming mutant protein has been implicated in a number of malignancies, including lung

adenocarcinoma (e.g., non-small cell), mucinous adenoma, ductal carcinoma of the pancreas, breast cancer, bladder cancer, thyroid cancer (including follicular and Hurthle cell tumors), gastric cancer, leukemias (including AML, primary plasma cell leukemia, and multiple myeloma), cholangiocarcinoma, and colon or rectal cancer. A cancer including cells having or harboring an activating KRAS mutation is a cancer associated with a KRAS mutation, as used herein.

KRAS is mutationally activated in 30-40% of rectal cancers by mutations at codons 12, 13, 22, 31, 61, 117, or 146 of SEQ ID NO: l . The significance oiKRAS mutational status for colorectal cancer is highlighted by the clinical data regarding the use of anti-EGFR therapy - potential benefit to anti-EGFR is restricted to KRAS wild- type (WT) patients (Jonker et al, The New England Journal of Medicine 357, 2040- 2048 (2007); Cunningham et al.,. The New England Journal of Medicine 351, 337- 345 (2004); Van Cutsem et al, J Clin Oncol 25, 1658-1664 (2007); Amado et al. J Clin Oncol 26, 1626-1634 (2008); Cengel et al, Neoplasia 9, 341-348 (2007); De Roock et al, Ann Oncol 19, 508-515 (2008); Di Fiore et al, Brit. J. Cancer 96, 1166- 1169 (2007); Freeman et al, Clin Colorectal Cancer 7, 184-190 (2008); Karapetis et al, The New England Journal of Medicine 359, 1757-1765 (2008); Lievre et al, J Clin Oncol 26, 374-379 (2008); Richman et al., J Clin Oncol 27, 5931-5937 (2009)). Thus, patients with metastatic colorectal cancer now routinely undergo KRAS mutational analysis. Further evidence supporting a biological difference is a recent analysis of 711 patients treated on the FOCUS trial demonstrating a worse overall survival in patients with KRAS mutation (HR 1.24, P=0.008) when treated with FOLFIRI or FOLFOX (Richman et al., J Clin Oncol 27:5931-5937 (2009)).

Mutant RAS has been implicated in radiation response; transfection of an activated RAS allele into rodent cells made them radioresistant (Cengel et al, Neoplasia 9, 341-348 (2007); Bernhard et al, Cancer Research 60, 6597-6600 (2000); Brunner et al, Cancer Research 65, 8433-8441 (2005); Brunner et al, Cancer Research 63, 5656-5668 (2003); Gupta et al., Cancer Research 61, 4278-4282 (2001); McKenna et al, Oncogene 22, 5866-5875 (2003)). In human colorectal cancer cell lines, genetic ablation of activated K-RAS restores radiosensitivity (Fig. 2) (McKenna et al, Oncogene 22, 5866-5875 (2003)). Limited reports have evaluated the impact of KRAS status on tumor regression with chemoradiation. For rectal cancer, a number of small studies have evaluated the impact of KRAS status on response to

chemoradiation. Bengala and colleagues conducted a study of 40 patients treated with cetuximab-based chemoradiation and analyzed the study by KRAS status (Bengala et al, Ann Oncol 2009;20:469-474). Of the 39 assessable patients, 30 had tumors that were KRAS wild type and 9 tumors were KRAS mutant. Eleven of 30 KRAS wild type tumors had a robust response to chemoradiation, with a tumor regression grade (TRG) 3-4, while only 1 of 9 KRAS mutant tumors had a TRG 3-4. Another cetuximab- based chemoradiation study (Debucquoy et al, J Clin Oncol 2009;27:2751-57) evaluated 41 patient tumors (-30% KRAS mutant) and showed a trend towards better response using Wheeler score (P=0.09). A study evaluating mutational status of genes implicated in colorectal cancer development also evaluated the impact of KRAS status on response to conventional chemoradiation (Zauber et al, Int J Radiat Oncol Biol Phys 2009;472:472-476). 53 patients were evaluated, of whom 13 (34%) had KRAS mutant tumors. Stage I (yp TO-2, NO) regression was seen in 49% of the KRAS wild-type tumors, compared with 33% of the KRAS mutant tumors. This result was not statistically significant, owing to the small numbers of patients evaluated.

Nevertheless, it is worth noting that, in all three studies, response was higher in KRAS wild type tumors than KRAS mutant tumors.

Methods of Treatment

Described herein are methods for the use of Midostaurin, or a

pharmaceutically acceptable salt thereof, for the treatment of a cancer associated with mutant KRAS, e.g., a radiation-resistant cancer associated with mutant KRAS, e.g., colorectal cancer associated with mutant KRAS. In some embodiments the cancer is rectal cancer. In some embodiments, the cancer is lung adenocarcinoma (e.g., non- small cell), mucinous adenoma, pancreatic cancer (e.g., ductal carcinoma of the pancreas), breast cancer, bladder cancer, thyroid cancer (including follicular and Hurthle cell tumors), gastric cancer, cholangiocarcinoma, or colorectal carcinoma.

In some embodiments, the methods for treating a radiation-resistant cancer associated with mutant KRAS in a subject include detecting the presence of cancer (e.g., tumor) cells having an activating KRAS mutation as known in the art and described herein; and administering Midostaurin in an amount that is therapeutically effective.

In some embodiments, the methods for treating a radiation-resistant cancer associated with mutant KRAS in a subject include detecting the presence of cancer (e.g., tumor) cells having an activating KRAS mutation as known in the art and described herein; administering Midostaurin in an amount that is therapeutically effective; and administering a therapeutically effective dose of radiation.

In some embodiments, the subject is a mammal, e.g., a human, who has or is suspected of having a radiation-resistant cancer associated with mutant KRAS.

As used in this context, to "treat" means to ameliorate at least one symptom of the radiation-resistant cancer associated with mutant KRAS. In some embodiments, the primary evaluation of antitumor effect is determined by pathological response, e.g., based on a known response rating system. In some embodiments, radiologic examination, e.g., using CT, MRI, x-ray, FDG PET, and/or FDG PET/CT can be used to detect a change in the size of a tumor (with shrinkage being a sign of successful treatment). For example, for rectal cancer, the Dworak Tumor Regression Grade (TRG) can be determined (Du et al, Nature biotechnology 27:77-83 (2009)). In addition the percentage of patients with pathological complete response (CR) will be determined.

Methods of Detecting KRAS Activating Mutations

A number of methods are known in the art for detecting activating mutations oiKRAS, and any method that detects activating mutations can be used. For example, direct sequencing of the gene, or relevant regions of the gene, can be used (e.g., those regions encompassing codons 12, 13, 22, 31, 61, 117, and/or 146). In some embodiments, next-gen sequencing methods can be used, e.g., as described in Mardis, Annu Rev Genomics Hum Genet. 9:387-402 (2008), Zhou et al, Sci China Life Sci. 53(l):44-57 (2010), and Wood et al, Sicence 318: 1108-1113 (2007). Alternatively, or in addition, allele-specific polymerase chain reaction, or assays that use direct hybridization technology, can also be used. For some mutations, e.g., mutation at codon 13, PCR-RFLP can be used (see, e.g., Shahrzad et al, Cancer Res.

65(18):8134-41 (2005); Shahrzad et al, Oncogene 27:3729-3738 (2008)).

In general, the detection of mutations is performed on DNA from cells known or suspected to be cancerous. In some embodiments, the DNA is from tumor sections, e.g., obtained from fresh frozen tumor tissue or tumor sections in a formalin-fixed, paraffin-embedded block. Commercially available assays can be used, e.g., Allele- specific PCR (DxS/Histogenex); direct sequencing (Gentris); allele-specific hybridization (Invitek); or allele-specific PCR extension (Genzyme).

For further descriptions of methods used to detect mutations, see, e.g., Edkins et al, Cancer Biol Ther. 5(8):928-32 (2006); Amado et al. J Clin Oncol 26, 1626-1634 (2008); Cengel et al, Neoplasia 9, 341-348 (2007); De Roock et al, Ann Oncol 19, 508-515 (2008); Di Fiore et al., Brit. J. Cancer 96, 1166-1169 (2007); Freeman et al, Clin Colorectal Cancer 7, 184-190 (2008); Karapetis et al, The New England Journal of Medicine 359, 1757-1765 (2008); Lievre et al, J Clin Oncol 26, 374-379 (2008); Richman et al, J Clin Oncol 27, 5931-5937 (2009); Bernhard et al, Cancer research 60, 6597-6600 (2000); and Brunner et al, Cancer research 65, 8433-8441 (2005), all of which are incorporated herein by reference in their entirety. Alternatively or in addition, the presence of mutant K-RAS proteins can be detected using an assay that detects K-RAS activity, e.g., by detecting the GTP- binding status of K-RAS.

Midostaurin (PKC412)

Midostaurin is N- [(9 S, 1 OR, 11 R, 13 R)-2,3 , 10, 11 , 12, 13 -hexahydro- 10-methoxy- 9-methyl-l -oxo-9- , 13-epoxy- lH,9H-diindolo[ 1 ,2,3-gh:3',2', 1 '-lm]pyrrolo[3,4- j][l,7]benzodiaz- onin-l l-yl]-N-methylbenzamide of the formula (I):

Formula I

or a salt thereof, hereinafter: "Compound of formula I or Midostaurin". The compound of formula I or Midostaurin [International Nonproprietary Name] is also known as PKC-412.

Midostaurin is a derivative of the naturally occurring alkaloid staurosporine, and has been specifically described in the European patent No. 0 296 110 published on Dec. 21, 1988, as well as in U.S. Pat. No. 5,093,330 published on Mar. 3, 1992, and Japanese Patent No. 2 708 047. See also U.S. PG Pub. No. 2009/0075972.

Midostaurin and its manufacturing process have been specifically described in many documents.

In general, in the methods described herein, Midostaurin can be administered parenterally, e.g., intraperitoneally, intravenously, intramuscularly, subcutaneously, intratumorally, or rectally, or enterally, e.g., orally. Previous phase I studies of midostaurin have yielded a maximum tolerated dose of about 150 mg/day, with undesirable side effects occurring more frequently at doses of 225 and 300 mg/day. See, e.g., Propper et al, J Clin Oncol 19: 1485-1492 (2001)

In some embodiments, the methods include administering a dose of about 150 mg/day. In some embodiments, the methods include administering the Midostaurin on an escalating dose schedule, e.g., of about 25-225 mg/M²/day. In some embodiments, the methods include administering the Midostaurin in multiple doses per day, e.g., 2-3 doses of about 50 mg.

An exemplary intravenous daily dosage is 0.1 to 10 mg/kg body weight or, for most larger primates, a daily dosage of 150-300 mg. A typical intravenous dosage is 3 to 5 mg/kg, three to seven times a week.

For example, Midostaurin can be administered orally in dosages up to about 150-200 mg/day, for example 100 to 150 mg/day. The Midostaurin can be administered as a single dose or split into two or three doses daily, preferably two doses. An exemplary dose is 150 mg/day, in particular 75 mg twice a day or 50 mg three times a day. The upper limit of dosage is that imposed by side effects and may be determined by trial for the patient being treated.

In some embodiments, the therapeutically effective amount of Midostaurin is administered to a mammal subject 7 to 4 times a week or about 100% to about 50% of the days in the time period, for a period of from one to six weeks, followed by a period of one to three weeks, wherein the agent is not administered and this cycle being repeated for from 1 to several cycles.

Usually, a small dose is administered initially and the dosage is gradually increased until the optimal dosage for the host under treatment is determined. The upper limit of dosage is that imposed by side effects and can be determined by trial for the host being treated.

In some embodiments, tumor cells from the subject are tested to evaluate whether a sufficient dose of Midostaurin is getting to the tumor. For example, detection of phospho-c-Jun levels could be used to determine whether a sufficient dosage has been administered to the tumor; a decrease in phospho-c-Jun levels in tumor cells) as compared to a reference (e.g., cells from the same tumor prior to treatment with Midostaurin) would indicate that a sufficient dose has been administered. As one example, if a subject does not respond, e.g., a subject who was otherwise expected to respond based on the presence of a K-ras mutant cancer, levels of phospho-c-Jun can be determined to see if the non-responder did not respond because the drug failed to reach the tumor in a sufficient amount. Any method known in the art for measuring levels of phospho-c-Jun can be used, e.g., western blot, or Luminex-based methods (as in Fig. 4).

Midostaurin may be combined with one or more pharmaceutically acceptable carriers and, optionally, one or more other conventional pharmaceutical adjuvants and administered enterally, e.g. orally, in the form of tablets, capsules, caplets, etc. or parenterally, e.g., intraperitoneally or intravenously, in the form of sterile injectable solutions or suspensions. The enteral and parenteral compositions may be prepared by conventional means. The parenteral (e.g., infusion) solutions according to the present invention are preferably sterile.

Midostaurin may be formulated into enteral and parenteral pharmaceutical compositions containing an amount of the active substance that is effective for treating the diseases and conditions named hereinbefore, such compositions in unit dosage form and such compositions comprising a pharmaceutically acceptable carrier.

Examples of useful compositions are described in the European patents No. 0 296 1 10, No. 0 657 164, No. 0 296 110, No. 0 733 372, No. 0 71 1 556, No. 0 711 557.

Some exemplary compositions are described in the European patent No. 0 657 164 published on Jun. 14, 1995. The described pharmaceutical compositions comprise a solution or dispersion of Midostaurin in a saturated polyalkylene glycol glyceride, in which the glycol glyceride is a mixture of glyceryl and polyethylene glycol esters of one or more Cs-Cis saturated fatty acids.

In some embodiments, Midostaurin is provided as 25 mg soft gelatin capsules. These capsules contain Polyoxyl 40 hydrogenated castor oil, Corn oil-mono-di- triglycerides, Macrogol 400 / Polyethylene glycol 400, Ethanol / Dehydrated alcohol, All-rac-a-Tocopherol / Vitamin E, Gelatin (porcine), Glycerol 85%, Titanium dioxide, Iron oxide red E172 and Iron oxide yellow E172.

Radiation Therapy

In some embodiments, the methods described herein include the

administration of radiation to tumor cells or tissue associated with mutant KRAS, e.g., determined to have a mutant KRAS gene or to express mutant K-RAS.

Methods of selecting and administering one or more doses of radiation are known in the art, administered using an external source or an internal source (e.g., an implanted source), or during surgical treatment. Taking rectal cancer as an example, the radiation therapy (RT) can be administered as external beam whole pelvic RT, intraoperative RT, and local RT, e.g., high-dose-rate endorectal brachytherapy (HDRBT). See, e.g., Hoffe et al, Cancer Control. 2010; 17(l):25-34 (2010), and references cited therein.

As one of skill in the art will appreciate, the dose of radiation to be administered will depend on a number of factors, including the radiation source; the size, type and location of the tumor; and the overall health of the subject. See, e.g., Perez and Brady's Principles and Practice of Radiation Oncology, Halperin et al, eds. Fifth Ed. (2008 Lippincott Williams & Wilkins); Cox and Ang, Radiation Oncology: Rationale, Technique, Results, (2003 Mosby Inc); and Colorectal Cancer: a Clinical Guide to Therapy. Bleiberg et al, eds. (2002 Martin Dunitz).

In some embodiments, the tumor is treated with 3-D conformal radiation therapy using shaped blocks to a dose of 45 Gy in 1.8 Gy/fraction. A boost of 5.4 Gy can be delivered to the tumor plus a 2 cm margin and to the presacral space.

Chemotherapy/Immunotherapy

The methods described herein can also be used in conjunction with administration of other treatments, e.g., chemotherapeutic agents or

immunotherapeutic agents, as are known in the art. The agents can be administered systemically, regionally, or locally to the cancer.

In some embodiments, the methods include administering one or more additional therapeutic agents to the subject. For example, the methods may include administering one or more chemotherapeutic agents to the subject, e.g., an antimetabolite, antitubulin, platinum-containing agent, or other agents, e.g., a topoisomerase 1 inhibitor, e.g., irinotecan.

An "antimetabolite" as used herein is a chemical with a similar structure to a substance (a metabolite) required for normal biochemical reactions, yet different enough to interfere with the normal functions of cells. Antimetabolites include purine and pyrimidine analogs that interfere with DNA synthesis. Exemplary

antimetabolites include, e.g., aminopterin, 2 -chlorodeoxy adenosine, cytosine arabinoside (ara C), cytarabine, fludarabine, fluorouracil (5-FU) (and its derivatives, which include capecitabine and tegafur), gemcitabine, methopterin, methotrexate, pemetrexed, raltitrexed, trimetrexate, 6-mercaptopurine, and 6-thioguanine. An "antitubulin" as used herein refers to a chemotherapeutic agent that blocks cell division by inhibiting the mitotic spindle. Antitubulin agents include, for example, the taxanes paclitaxel and docetaxel, and the vinca alkaloids vinorelbine, vincristine, vinblastine, vinflunine, and vindesine.

A "platinum-containing agent" as used herein includes chemotherapeutic agents that contain platinum. Platinum-containing agents cross-link with and alkylate DNA, which results in the inhibition of DNA synthesis and transcription. The platinum-containing agents can act in any cell cycle, and consequently kill neoplastic as well as healthy dividing cells. Platinum-containing agents include, for example, cisplatin, carboplatin and oxaliplatin.

For rectal cancer, in some embodiments, the methods can include

administration of 5-FU, with or without leucovorin. 5-Fluorouracil (5-FU) is an antimetabolite that is systemically metabolized with the enzyme dihydropyrimidine dehydrogenase being rate limiting. It forms the backbone of treatment for most gastrointestinal malignancies, and is a standard component of chemoradiation in locally advanced rectal cancer. 5-FU is used in combination with radiation in many types of cancer due to its radiation potentiation qualities. The usual dose of 5-FU when administered by continuous infusion with radiation therapy is 225 mg/M²/d. In some embodiments, the methods described herein include administration of 150 mg/day Midostaurin in combination with a continuous infusion of 200 mg/M²/d 5-FU.

For colon cancer, in some embodiments, the methods can include the administration of 5-FU, with or without one or more of leucovorin, irinotecan (Camptosar) and oxaliplatin (Eloxatin). Using common terminology, treatment that includes 5-FU, leucovorin, and irinotecan is referred to as "FOLFIRI" while treatment with 5-FU, leucovorin, and oxaliplatin is called "FOLFOX." These agents can be administered before, during, or after radiation therapy or surgical or ablative therapy. In some embodiments, the 5-FU is administered continuously, e.g., using an infusion pump. In some embodiments, the 5-FU is administered as a bolus, e.g., when used in combination with one the other agents. In some embodiments, when 5-FU is given, leucovorin will also be administered. See, e.g., O'Connell, Oncology (Williston Park). 18(6):751-5; 755-8 (2004).

In some embodiments, the methods also include administration of an immunotherapeutic agent, e.g., a monoclonal antibody such as bevacizumab (Avastin) (a humanized monoclonal antibody against vascular endothelial growth factor). Other anti-cancer antibodies are known in the art, and a skilled practitioner could readily select an appropriate agent for administration.

Ablative or Surgical Treatment

The methods described herein can also be used in conjunction with ablative or surgical excision of the cancer cells. Ablative treatment can include any method that causes removal of cancerous cells or tissues, e.g., laser therapy, phototherapy, radiofrequency ablation, and cryotherapy. Surgical treatments can include invasive and non-invasive excision. Taking colon cancer as an example, surgical treatment can include polypectomy, partial resection and anastomosis, resection and colostomy. These surgical and ablative methods can be performed before, during, and/or after treatment with Midostaurin and radiation.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1. Midostaurin Induces Apoptosis in Cells Expressing Mutant K-RAS

In an effort to understand the oncogenic phenotype of mutant K-RAS, a small molecule screen was performed to identify compounds that can affect the overall viability of cells expressing mutant K-RAS. This screen utilized DLD-1 colon cancer cells, which express K-RAS^G13D, and DKs-8, an isogenic derivative of DLD-1 that expresses only wild-type K-RAS. Both DLD-1 and DKs-8 are also mutant for ^ C,

TRP53, and PIK3CA.

The assay consists of growing cells in 96-well format for 72 hours. Individual wells are either mock treated (with DMSO) or else exposed to a given small molecule at 0.01, 0.1, or 1 μΜ. Each treatment is done in eight replicates so that a single cell line occupies 4 rows of a 96-well plate. After 72 hours of growth, cells are fixed in

4% paraformaldehyde and stained with SYTO60 (Molecular Probes/Invitrogen), an infrared nucleic acid dye. Plates are scanned on a LiCor Odyssey Infrared Imaging

System. The amount of infrared emission from each well is directly proportional to the number of cells within that well, allowing us to quantify relative cell growth over the 72 hour period. After scanning, the quantitative data is analyzed to produce a curve that plots growth as a function of drug concentration (e.g., FIG. 1A). Non- parametric statistics are performed to determine whether significant differences exist between the curves for each of the cell lines.

In the course of the screen, it was found that DLD-1 cells are hypersensitive to Midostaurin, a broad-spectrum kinase inhibitor (FIG. 1A). Midostaurin induces a dose-dependent apoptotic response in colorectal cancer cells, with DLD-1 cells being hypersensitive to Midostaurin-induced cell death compared to DKs-8 cells (FIG. IB).

Example 2. Midostaurin Reverses the Radiation Resistance Associated with

Expression of Mutant K-RAS

The loss of mutant K-RAS affects many of the properties of DLD- 1 cells, for example growth in soft agar and subcutaneous growth in immunocompromised mice (Shirasawa et al, Science (New York, N.Y 260, 85-88 (1993)). The effect of loss of mutant KRAS on sensitivity to ionizing radiation was assessed by clonogenic survival assay.

Briefly, clonogenic survival was determined at radiation doses from 1 to 6 Gy. Cells from logarithmically growing cultures were plated at low density (about 5,000 or 10,000 per well) into 6 well plates, allowed to attach overnight, and then irradiated using a Cesium-137 source. Cells were treated with drugs 5-10 minutes before irradiation. Colonies (of greater than 50 cells) were stained with Crystal violet 14-21 days after irradiation. The surviving fraction at a given dose is defined as: number of colonies formed/(number of cells plated)X(plating efficiency). Each point on the survival curves represents the mean surviving fraction from at least three wells.

DKs-8 cells lacking K-RAS^G13D exhibit enhanced sensitivity to ionizing radiation (Fig. 2A). Similar to its ability to promote apoptosis in DLD-1 cells, Midostaurin can abrogate the radiation resistance associated with mutant K-RAS (FIG. 2A). In most cases, cancer patients receive radiation and chemotherapy prior to surgical resection. To determine whether Midostaurin might cooperate with chemotherapy to affect radiation response, clonogenic survival of DLD- 1 cells exposed to radiation was measured in the presence of Midostaurin and/or 5- fluorouracil (5-FU). Although 5-FU alone did not enhance the effect of radiation on DLD-1 cells, it cooperated with Midostaurin to decrease survival after exposure to radiation (FIG. 2B).

Taken together, these studies suggest that Midostaurin may serve as an effective radiosensitizer in rectal cancers expressing mutant K-RAS. Example 3. Exemplary Treatment Plan

Subjects with cancer expressing mutant K-RAS, e.g., rectal cancer expressing mutant K-RAS, are treated as follows with Midostaurin and 5-FU, e.g., administered on the following schedule:

Midostaurin is administered orally, beginning on day #1 of 5-FU and radiation. It is taken prior to radiation each day, on the following dose-escalation schedule:

5-FU is administered at a dose of 225 mg/M²/d by continuous infusion ambulatory infusion pump, 5 days a week, during the course of radiation. Example 4. Phospho-c-Jun as a Biomarker for Midostaurin Response

In this experiment, wild-type and mutant colorectal cancer cells were treated with Midostaurin (500 nM) for 1 hour. Following treatment, protein lysates were generated and the phosphorylation state of c-Jun on serine 63 (Ser63) was analyzed via Bio-Plex. K-Ras mutant cells have higher basal levels of phospho-c-Jun than wild-type cells. Moreover, as shown in Fig. 4, upon treatment with Midostaurin, both wild-type and mutant cells shows a dramatic reduction in phospho-c-Jun levels. This result suggests that phospho-c-Jun is a useful biomarker for Midostaurin response.

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 increasing sensitivity of a mutant KRAS associated cancer to

radiation, the method comprising:

identifying a cancer cell having a mutant KRAS gene;

administering a therapeutically effective dose of Midostaurin to the cell; and administering a therapeutically effective dose of radiation to the cell,

thereby increasing the sensitivity of the cell to radiation therapy.

2. A method of treating a subject who has a mutant KRAS associated cancer, the method comprising:

identifying a subject who has a cancer that is associated with a mutant KRAS gene; administering a therapeutically effective dose of Midostaurin to the subject; and administering a therapeutically effective dose of radiation to the subject, thereby treating the subject.

3. A method of selecting a treatment for a subject who has cancer, the method

comprising:

providing a sample comprising a cancer cell from the subject;

detecting the presence of mutant KRAS in the cell; and

selecting a treatment comprising administering Midostaurin and radiation for the subject if the cell has a mutant KRAS.

4. The method of claim 3, further comprising administering the treatment to the subject.

5. A method of treating a subject who has a mutant KRAS associated cancer, the method comprising:

identifying a subject who has a cancer that is associated with a mutant KRAS gene; administering a therapeutically effective dose of Midostaurin to the subject;

thereby treating the subject.

6. A method of selecting a treatment for a subject who has cancer, the method

comprising:

providing a sample comprising a cancer cell from the subject;

detecting the presence of mutant KRAS in the cell; and selecting a treatment comprising administering Midostaurin for the subject if the cell has a mutant KRAS.

7. The method of claim 6, further comprising administering the treatment to the subject.

8. The method of claims 1, 2, and 5, wherein the identifying step comprises

obtaining a cell of the cancer and detecting the presence of a mutant KRAS in the cell.

9. The method of claims 3 and 6, wherein detecting the presence of a mutant KRAS comprises detecting expression of a mutant K-RAS protein, or presence of a mutant KRAS nucleic acid.

10. The method of claim 9, wherein detecting presence of a mutant KRAS nucleic acid comprises detecting presence of a KRAS transcript or gene comprising an activating mutation.

1 1. The method of claim 10, wherein the activating mutation comprises an amino acid substitution of residue G12, G13, Q22, E31, Q61, K117or A146 of the mature protein.

12. The method of claims 1-11 further comprising administering a chemotherapeutic agent.

13. The method of claim 12, wherein the chemotherapeutic agent is 5-flurouracil (5- FU).

14. Use of Midostaurin in the treatment of a KRAS-mutation associated cancer.

15. The method or use of any of claims 1-14, wherein the cancer is selected from the group consisting of lung adenocarcinoma, mucinous adenoma, pancreatic cancer, breast cancer, bladder cancer, thyroid cancer, gastric cancer, cholangiocarcinoma, or colorectal carcinoma.

16. The method or use of any of claims 1-14, wherein the cancer is rectal carcinoma.