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Colorectal cancers harboring KRAS or BRAF mutations are refractory to current targeted therapies. Using data from a high-throughput drug screen, we have developed a novel therapeutic strategy that targets the apoptotic machinery using the BCL-2 family inhibitor ABT-263 (navitoclax) in combination with a TORC1/2 inhibitor, AZD8055. This combination leads to efficient apoptosis specifically in KRAS- and BRAF-mutant but not wild-type (WT) colorectal cancer cells. This specific susceptibility results from TORC1/2 inhibition leading to suppression of MCL-1 expression in mutant, but not WT, colorectal cancers, leading to abrogation of BIM/MCL-1 complexes. This combination strategy leads to tumor regressions in both KRAS-mutant colorectal cancer xenograft and genetically engineered mouse models of colorectal cancer, but not in the corresponding KRAS-WT colorectal cancer models. These data suggest that the combination of BCL-2/BCL-XL inhibitors with TORC1/2 inhibitors constitutes a promising targeted therapy strategy to treat these recalcitrant cancers. Significance: Effective targeted therapies directed against colorectal cancer with activating mutations in KRAS remain elusive. We have leveraged drug-screen data from a large panel of human colorectal cancers to uncover an effective, rational targeted therapy strategy that has preferential activity in colorectal cancers with KRAS or BRAF mutations. This combination may be developed for clinical testing. Cancer Discov; 4(1); 42–52. ©2013 AACR. See related commentary by Russo et al., p. 19 This article is highlighted in the In This Issue feature, p. 1

Purpose: BRAFV600E mutations are associated with poor clinical prognosis in colorectal cancer (CRC). Although selective BRAF inhibitors are effective for treatment of melanoma, comparable efforts in CRC have been disappointing. Here, we investigated potential mechanisms underlying this resistance to BRAF inhibitors in BRAFV600E CRC. Experimental Design: We examined phosphoinositide 3-kinase (PI3K)/mTOR signaling in BRAFV600E CRC cell lines after BRAF inhibition and cell viability and apoptosis after combined BRAF and PI3K/mTOR inhibition. We assessed the efficacy of in vivo combination treatment using a novel genetically engineered mouse model (GEMM) for BRAFV600E CRC. Results: Western blot analysis revealed sustained PI3K/mTOR signaling upon BRAF inhibition. Our BRAFV600E GEMM presented with sessile serrated adenomas/polyps, as seen in humans. Combination treatment in vivo resulted in induction of apoptosis and tumor regression. Conclusions: We have established a novel GEMM to interrogate BRAFV600E CRC biology and identify more efficacious treatment strategies. Combination BRAF and PI3K/mTOR inhibitor treatment should be explored in clinical trials. Clin Cancer Res; 19(10); 2688–98. ©2013 AACR.

Hereditary fructose intolerance (HFI) is a potentially fatal inherited metabolic disease caused by a deficiency of aldolase B activity in the liver and kidney. Over 40 disease-causing mutations are known in the protein-coding region of ALDOB. Mutations upstream of the protein-coding portion of ALDOB are reported here for the first time. DNA sequence analysis of 61 HFI patients revealed single base mutations in the promoter, intronic enhancer, and the first exon, which is entirely untranslated. One mutation, g.–132G>A, is located within the promoter at an evolutionarily conserved nucleotide within a transcription factor-binding site. A second mutation, IVS1+1G>C, is at the donor splice site of the first exon. In vitro electrophoretic mobility shift assays show a decrease in nuclear extract-protein binding at the g.–132G>A mutant site. The promoter mutation results in decreased transcription using luciferase reporter plasmids. Analysis of cDNA from cells transfected with plasmids harboring the IVS1+1G>C mutation results in aberrant splicing leading to complete retention of the first intron (~ 5 kb). The IVS1+1G>C splicing mutation results in loss of luciferase activity from a reporter plasmid. These novel mutations in ALDOB represent 2% of alleles in American HFI patients, with IVS1+1G>C representing a significantly higher allele frequency (6%) among HFI patients of Hispanic and African-American ethnicity.

Effective treatment options for advanced colorectal cancer (CRC) are limited, survival rates are poor, and this disease continues to be a leading cause of cancer-related deaths worldwide. Despite being a highly heterogeneous disease, a significant subset of patients with sporadic CRC typically harbor relatively few established 'driver' lesions. Here, we describe a collection of genetically engineered mouse models (GEMM) of sporadic CRC that combine lesions frequently altered in human patients, including well-characterized tumor suppressors and activators of MAPK signaling. Primary tumors from these models were profiled, and individual GEMM tumors segregated into groups based on their genotypes. Unique allelic and genotypic expression signatures were generated from these GEMMs and applied to clinically annotated human CRC patient samples. We provide evidence that a Kras signature derived from these GEMMs is capable of distinguishing KRAS mutant patients, and tracks with poor prognosis in two independent human patient cohorts. Further, the analysis of a panel of human CRC cell lines suggests that high expression of the GEMM Kras signature correlates with sensitivity to targeted pathway inhibitors. Together, these findings implicate GEMMs as powerful preclinical tools with the capacity to recapitulate relevant human disease biology, and support the use of genetic signatures generated in these models to facilitate future drug discovery and validation efforts.

Purpose: Effective therapies for KRAS-mutant colorectal cancer (CRC) are a critical unmet clinical need. Previously, we described genetically engineered mouse models (GEMM) for sporadic Kras-mutant and non-mutant CRC suitable for preclinical evaluation of experimental therapeutics. To accelerate drug discovery and validation, we sought to derive low-passage cell lines from GEMM Kras-mutant and wild-type tumors for in vitro screening and transplantation into the native colonic environment of immunocompetent mice for in vivo validation. Experimental Design: Cell lines were derived from Kras-mutant and non-mutant GEMM tumors under defined media conditions. Growth kinetics, phosphoproteomes, transcriptomes, drug sensitivity, and metabolism were examined. Cell lines were implanted in mice and monitored for in vivo tumor analysis. Results: Kras-mutant cell lines displayed increased proliferation, mitogen-activated protein kinase signaling, and phosphoinositide-3 kinase signaling. Microarray analysis identified significant overlap with human CRC-related gene signatures, including KRAS-mutant and metastatic CRC. Further analyses revealed enrichment for numerous disease-relevant biologic pathways, including glucose metabolism. Functional assessment in vitro and in vivo validated this finding and highlighted the dependence of Kras-mutant CRC on oncogenic signaling and on aerobic glycolysis. Conclusions: We have successfully characterized a novel GEMM-derived orthotopic transplant model of human KRAS-mutant CRC. This approach combines in vitro screening capability using low-passage cell lines that recapitulate human CRC and potential for rapid in vivo validation using cell line-derived tumors that develop in the colonic microenvironment of immunocompetent animals. Taken together, this platform is a clear advancement in preclinical CRC models for comprehensive drug discovery and validation efforts. Clin Cancer Res; 19(11); 2929–40. ©2013 AACR.

BRAF mutations occur in 10% to 15% of colorectal cancers and confer adverse outcome in the metastatic setting. Although RAF inhibitors such as vemurafenib (PLX4032) have proven effective in the treatment of BRAF-mutant melanoma, they are surprisingly ineffective in BRAF-mutant colorectal cancers, and the reason for this disparity remains unclear. Compared with BRAF-mutant melanoma cells, BRAF-mutant colorectal cancer cells were less sensitive to vemurafenib, and phospho-extracellular signal-regulated kinase (P-ERK) suppression was not sustained in response to treatment. Although transient inhibition of P-ERK by vemurafenib was observed in colorectal cancer, rapid ERK reactivation occurred through epidermal growth factor receptor (EGFR)-mediated activation of RAS and CRAF. BRAF-mutant colorectal cancers expressed greater levels of phospho-EGFR than BRAF-mutant melanomas, suggesting that colorectal cancers are specifically poised for EGFR-mediated resistance. Combined RAF and EGFR inhibition blocked reactivation of mitogen-activated protein kinase (MAPK) signaling in BRAF-mutant colorectal cancer cells and markedly improved efficacy in vitro and in vivo. These findings support the evaluation of combined RAF and EGFR inhibition in patients with BRAF-mutant colorectal cancer. Significance: BRAF valine 600 (V600) mutations occur in 10% to 15% of colorectal cancers, yet these tumors show a surprisingly low clinical response rate (∼5%) to selective RAF inhibitors such as vemurafenib, which have produced dramatic response rates (60%–80%) in melanomas harboring the identical BRAF V600 mutation. We found that EGFR-mediated MAPK pathway reactivation leads to resistance to vemurafenib in BRAF-mutant colorectal cancers and that combined RAF and EGFR inhibition can lead to sustained MAPK pathway suppression and improved efficacy in vitro and in tumor xenografts. Cancer Discovery; 2(3); 227–35. ©2012 AACR. This article is highlighted in the In This Issue feature, p. 193

Purpose To examine the in vitro and in vivo efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type colorectal cancer (CRC). Experimental Design PIK3CA mutant and wild-type human CRC cell lines were treated in vitro with NVP-BEZ235, and the resulting effects on proliferation, apoptosis, and signaling were assessed. Colonic tumors from a genetically engineered mouse (GEM) model for sporadic wild-type PIK3CA CRC were treated in vivo with NVP-BEZ235. The resulting effects on macroscopic tumor growth/regression, proliferation, apoptosis, angiogenesis, and signaling were examined. Results In vitro treatment of CRC cell lines with NVP-BEZ235 resulted in transient PI3K blockade, sustained decreases in mTORC1/mTORC2 signaling, and a corresponding decrease in cell viability (median IC50 = 9.0–14.3 nM). Similar effects were seen in paired isogenic CRC cell lines that differed only in the presence or absence of an activating PIK3CA mutant allele. In vivo treatment of colonic tumor-bearing mice with NVP-BEZ235 resulted in transient PI3K inhibition and sustained blockade of mTORC1/mTORC2 signaling. Longitudinal tumor surveillance by optical colonoscopy demonstrated a 97% increase in tumor size in control mice (p = 0.01) vs. a 43% decrease (p = 0.008) in treated mice. Ex vivo analysis of the NVP-BEZ235-treated tumors demonstrated a 56% decrease in proliferation (p = 0.003), no effects on apoptosis, and a 75% reduction in angiogenesis (p = 0.013). Conclusions These studies provide the preclinical rationale for studies examining the efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type CRC.

Mutations in the aldolase B gene (ALDOB) impairing enzyme activity toward fructose-1-phosphate cleavage cause hereditary fructose intolerance (HFI). Diagnosis of the disease is possible by identifying known mutant ALDOB alleles in suspected patients; however, the frequencies of mutant alleles can differ by population. Here, 153 American HFI patients with 268 independent alleles were analyzed to identify the prevalence of seven known HFI-causing alleles (A149P, A174D, N334K, Delta4E4, R59Op, A337V, and L256P) in this population. Allele-specific oligonucleotide hybridization analysis was performed on polymerase chain reaction (PCR)-amplified genomic DNA from these patients. In the American population, the missense mutations A149P and A174D are the two most common alleles, with frequencies of 44% and 9%, respectively. In addition, the nonsense mutations Delta4E4 and R59Op are the next most common alleles, with each having a frequency of 4%. Together, the frequencies of all seven alleles make up 65% of HFI-causing alleles in this population. Worldwide, these same alleles make up 82% of HFI-causing mutations. This difference indicates that screening for common HFI alleles is more difficult in the American population. Nevertheless, a genetic screen for diagnosing HFI in America can be improved by including all seven alleles studied here. Lastly, identification of HFI patients presenting with classic symptoms and who have homozygous null genotypes indicates that aldolase B is not required for proper development or metabolic maintenance.

BRAFmutations occur in ∼10% of colorectal cancers (CRCs) and confer poor prognosis. While RAF inhibitor monotherapy leads to response rates of ∼60% in BRAF mutant melanoma, response rates in BRAF mutant CRC are disappointingly low (∼5%), suggesting a fundamental difference between these tumor types, despite the mutual presence of a BRAF V600 mutation. Previously, our group and others found that while RAF inhibitors lead to profound and sustained suppression of MAPK signaling in BRAF mutant melanoma cells, suppression of MAPK signaling by RAF inhibitors in BRAF mutant CRC cells is transient, and the MAPK pathway is rapidly reactivated despite the continued presence of drug. These data suggest that MAPK pathway suppression by RAF inhibitors alone may be inadequate in BRAF mutant CRC and that combinations of RAF inhibitors with other targeted agents might be required to achieve robust MAPK suppression and clinical responses. More recently, our group and others found that in many BRAF mutant CRCs, MAPK pathway reactivation is driven by feedback signaling through EGFR via RAS and CRAF. Importantly, we found that the combination of a RAF inhibitor and an EGFR inhibitor can lead to sustained MAPK suppression and improved efficacy, leading to marked tumor regressions in preclinical BRAF mutant CRC xenograft models. These data have led to clinical trials in BRAF mutant CRC patients evaluating RAF inhibitor combinations, including combinations of RAF+EGFR, RAF+MEK, and RAF+MEK+EGFR inhibitors. Initial data from these trials, reported at ASCO 2014, have shown encouraging efficacy, with some trials showing initial response rates of as much as 40%. However, as with all targeted therapies, clinical benefit is invariably limited by the emergence of acquired resistance to these therapies. In this study, we sought to identify clinically relevant mechanisms of acquired resistance to RAF inhibitor combinations in BRAF mutant CRC, in order to understand the signaling changes leading to resistance and to devise therapeutic strategies to overcome or prevent resistance. To do so, we obtained tumor biopsies from BRAF mutant CRC patients upon disease progression, after initial response or prolonged stable disease on RAF+EGFR, RAF+MEK, or RAF+MEK+EGFR inhibitor combinations. Matched pre-treatment, post-progression, and normal DNA were analyzed by whole exome sequencing (WES) and RNA sequencing. To supplement this approach, in vitro modeling of acquired resistance was performed in BRAF mutant CRC cell lines. In one BRAF mutant CRC patient with prolonged stable disease on a RAF+EGFR inhibitor combination, WES identified KRAS amplification in a progressing lesion. RNA sequencing confirmed KRAS transcript overexpression, and KRAS amplification (∼25-fold) was confirmed by FISH in the post-progression biopsy, but was absent in a pre-treatment biopsy. Interestingly, in resistant clones generated from BRAF mutant CRC cell lines selected with either RAF+EGFR or RAF+MEK inhibitor combinations, KRAS exon 2 mutations were identified. Either KRAS amplification or KRAS mutation led to sustained MAPK pathway activity and cross-resistance to either RAF+EGFR or RAF+MEK inhibitor combinations. However, an ERK inhibitor, either alone or in combination with a RAF inhibitor retained the ability to suppress the MAPK pathway and could overcome resistance. In a second patient with a minor response to a RAF+EGFR inhibitor combination, BRAF amplification was identified in a progressing lesion, which was confirmed by FISH and was not present in a pre-treatment biopsy of the same lesion. Previously, our laboratory found that amplification of mutant BRAF could cause resistance to RAF or MEK inhibitors in BRAF mutant CRC cells by abrogating the ability of these inhibitors to suppress MAPK signaling. Importantly, an ERK inhibitor, either alone or in combination with a RAF inhibitor could suppress MAPK signaling and overcome resistance in this setting. In one patient with a minor response to a RAF+MEK inhibitor combination, WES identified the presence of an ARAF Q489L mutation and a MEK1 F53L mutation in a single progressing lesion, suggesting possible intra-lesional heterogeneity of acquired resistance mechanisms. However, utilizing a cell line derived from the patient's post-progression biopsy, we found that 30 out of 30 single cell clones harbored both the ARAF and MEK1 mutations, and that the MEK1 F53L seemed to function as the primary driver of acquired resistance in these resistant tumor cells. MEK1 F53L expression markedly abrogated the ability of RAF+MEK and RAF+EGFR inhibitor combinations to suppress MAPK signaling. Interestingly, an ERK inhibitor was able to effectively suppress MAPK signaling and overcome resistance. In a second patient with a minor response to RAF+MEK, WES identified a focal amplicon on chromosome 2 in a progressing lesion, encompassing the c-mer oncogene (MERTK) receptor tyrosine kinase and the MAPK-regulated checkpoint kinase BUB1. Post-progression biopsies from two patients who progressed after prolonged stable disease to the triple combination of a RAF+MEK+EGFR inhibitor combination are currently being analyzed by WES, and updated results will be presented. In summary, RAF inhibitor combinations are leading to promising initial response rates in recent clinical trials in BRAF mutant CRC patients. The frequent identification of alterations leading to reactivation of MAPK pathway signaling upon clinical acquired resistance to RAF+EGFR or RAF+MEK combinations underscores the MAPK pathway as a valid and critical target in BRAF mutant CRC. Importantly, we found that many of these resistance mechanisms could be overcome by an ERK inhibitor or ERK inhibitor-based combinations, suggesting that ERK inhibitors may be key components of therapeutic combinations to be explored in future clinical trials for BRAF mutant CRC. Further efforts to understand acquired resistance mechanisms will be vital to developing novel therapeutic strategies to overcome resistance and extend clinical benefit in this lethal CRC subtype. Citation Format: Ryan B. Corcoran, Leanne G. Ahronian, Eliezer Van Allen, Erin M. Coffee, Nikhil Wagle, Eunice L. Kwak, Jason E. Faris, A. John Iafrate, Levi A. Garraway, Jeffrey A. Engelman. Clinical acquired resistance to RAF inhibitor combinations in BRAF mutant colorectal cancer through MAPK pathway alterations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr NG04. doi:10.1158/1538-7445.AM2015-NG04

Although KRAS is the most commonly mutated oncogene in human cancer, KRAS has proven difficult to target pharmacologically, and no effective therapies exist for KRAS-mutant cancers. Recently, there has been evidence that targeted therapy combinations inhibiting multiple downstream effectors of KRAS may be a promising approach for KRAS-mutant cancers. We developed a pooled shRNA-drug screen strategy to identify genes that, when inhibited, cooperate with MEK inhibitors to kill KRAS-mutant cancer cells. The anti-apoptotic BH3 family gene BCL-XL emerged as a top hit through this approach. ABT-263 (navitoclax), a chemical inhibitor that blocks the ability of BCL-XL to bind and inhibit pro-apoptotic proteins, in combination with a MEK inhibitor led to dramatic apoptosis in the vast majority of KRAS-mutant cell lines tested from different tissue types. Mechanistic studies revealed that MEK inhibition led to marked induction of the pro-apoptotic protein BIM in KRAS-mutant cancer cells, but that BIM remained bound and inhibited by BCL-XL. Pharmacologic inhibition of BCL-XL with ABT-263 disrupted this inhibitory complex, allowing BIM to trigger apoptosis. Epithelial differentiation and E-cadherin expression correlated with increased sensitivity to this inhibitor combination across a panel of 30 KRAS-mutant cell lines, while epithelial-to-mesenchymal transition (EMT) correlated with resistance. This combination also caused marked in vivo tumor regressions in three independent KRAS-mutant xenografts and in established lung tumors in two genetically-engineered KRAS-driven lung cancer mouse models. These data support combined BCL-XL/MEK inhibition as a promising therapeutic approach for evaluation in future clinical trials for patients with KRAS-mutant cancers. This abstract is also presented as Poster B19. Citation Format: Ryan B. Corcoran, Katherine A. Cheng, Aaron N. Hata, Anthony C. Faber, Hiromichi Ebi, Erin M. Coffee, Patricia Greninger, Ronald D. Brown, Jason T. Godfrey, Travis J. Cohoon, Youngchul Song, Eugene Lifshits, Toshi Shioda, Dora Dias-Santagata, Anurag Singh, Jeffrey Settleman, Cyril H. Benes, Mari Mino-Kenudson, Kwok-Kin Wong, Jeffrey A. Engelman. Synthetic lethal interaction of combined BCL-XL and MEK inhibition promotes tumor regressions in KRAS-mutant cancer models. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr PR09.

SummaryKRAS is the most commonly mutated oncogene, yet no effective targeted therapies exist for KRAS mutant cancers. We developed a pooled shRNA-drug screen strategy to identify genes that, when inhibited, cooperate with MEK inhibitors to effectively treat KRAS mutant cancer cells. The anti-apoptotic BH3 family gene BCL-XL emerged as a top hit through this approach. ABT-263 (navitoclax), a chemical inhibitor that blocks the ability of BCL-XL to bind and inhibit pro-apoptotic proteins, in combination with a MEK inhibitor led to dramatic apoptosis in many KRAS mutant cell lines from different tissue types. This combination caused marked in vivo tumor regressions in KRAS mutant xenografts and in a genetically engineered KRAS-driven lung cancer mouse model, supporting combined BCL-XL/MEK inhibition as a potential therapeutic approach for KRAS mutant cancers.