KRAS Mutant Tumor Growth Research Articles

Abstract 1955: Inhibition of eIF4A by eFT226 blocks KRAS mutant tumor growth

Abstract Mutations in KRAS are among the most common oncogenic lesions across a variety of human cancers. Activation of KRAS directs signaling via the MAPK and PI3K pathways to promote tumor growth. One outcome of enhanced KRAS signaling is the induction of mRNA translation by eIF4A, eIF4E, and eIF4G, which together comprise the eIF4F complex. eIF4A is an RNA helicase that functions to unwind elements in the 5'-untranslated region (UTR) of mRNAs to facilitate scanning of the 40S ribosomal subunit. eFT226 is a highly potent and selective inhibitor of eIF4A that functions by forming a ternary complex between eIF4A, eFT226 and specific polypurine motifs in the 5'-UTR of select mRNAs, thus blocking ribosome scanning and inhibiting mRNA translation. This polypurine sequence motif is highly enriched in the 5'-UTR of eFT226 target genes, many of which are known proto-oncogenes. Translational profiling revealed KRAS to be a target of eFT226 and 5'-UTR sequence analysis of KRAS mRNA identified the polypurine regulatory motif, which imparts sensitivity to eFT226 mediated inhibition of translation. In cell-based reporter assays used to monitor translation, mutation of the KRAS 5'-UTR polypurine motifs resulted in a 10-fold decrease in sensitivity to eFT226 relative to the wild-type sequence. In a cell panel screen for in vitro apoptosis induction by eFT226, the most sensitive models were enriched for cell lines driven by KRAS mutations. Consistent with these results, treatment with eFT226 decreased KRAS protein levels, repressed downstream MAPK signaling, inhibited cell proliferation, and induced apoptosis in a collection of non-small cell lung, colorectal and pancreatic KRAS mutant cancer cell lines. The ability of eFT226 to block tumor cell growth and induce apoptosis is independent of the specific KRAS mutation present (e.g. G12C/V, G13D, Q61H/L) suggesting that eFT226 could be broadly efficacious in treating tumors with activating KRAS mutations. Treatment of KRAS mutant solid tumor xenografts with eFT226 as a monotherapy significantly inhibited tumor growth. Together, these results highlight the ability of eFT226 to inhibit growth and promote apoptosis in KRAS mutant tumors and support the clinical development of eFT226 in KRAS driven tumors. A clinical trial evaluating eFT226 in patients with solid tumor malignancies has initiated. Citation Format: Craig R. Stumpf, Vikas K. Goel, Joan Chen, Jocelyn Staunton, Emily M. Santori, Maria Barrera, Haleigh Howard, Kevin R. Webster, Gary G. Chiang, Peggy A. Thompson. Inhibition of eIF4A by eFT226 blocks KRAS mutant tumor growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1955.

A Role for Mitochondrial Translation in Promotion of Viability in K-Ras Mutant Cells

Activating mutations in the KRAS oncogene are highly prevalent in tumors, especially those of the colon, lung, and pancreas. To better understand the genetic dependencies that K-Ras mutant cells rely upon for their growth, we employed whole-genome CRISPR loss-of-function screens in two isogenic pairs of cell lines. Since loss of essential genes is uniformly toxic in CRISPR-based screens, we also developed a small hairpin RNA (shRNA) library targeting essential genes. These approaches uncovered a large set of proteins whose loss results in the selective reduction of K-Ras mutant cell growth. Pathway analysis revealed that many of these genes function in the mitochondria. For validation, we generated isogenic pairs of cell lines using CRISPR-based genome engineering, which confirmed the dependency of K-Ras mutant cells on these mitochondrial pathways. Finally, we found that mitochondrial inhibitors reduce the growth of K-Ras mutant tumors invivo, aiding in the advancement of strategies to target K-Ras-driven malignancy.

Macroautophagy is dispensable for growth of KRAS mutant tumors and chloroquine efficacy

Macroautophagy is a key stress-response pathway that can suppress or promote tumorigenesis depending on the cellular context. Notably, Kirsten rat sarcoma (KRAS)-driven tumors have been reported to rely on macroautophagy for growth and survival, suggesting a potential therapeutic approach of using autophagy inhibitors based on genetic stratification. In this study, we evaluated whether KRAS mutation status can predict the efficacy to macroautophagy inhibition. By profiling 47 cell lines with pharmacological and genetic loss-of-function tools, we were unable to confirm that KRAS-driven tumor lines require macroautophagy for growth. Deletion of autophagy-related 7 (ATG7) by genome editing completely blocked macroautophagy in several tumor lines with oncogenic mutations in KRAS but did not inhibit cell proliferation in vitro or tumorigenesis in vivo. Furthermore, ATG7 knockout did not sensitize cells to irradiation or to several anticancer agents tested. Interestingly, ATG7-deficient and -proficient cells were equally sensitive to the antiproliferative effect of chloroquine, a lysosomotropic agent often used as a pharmacological tool to evaluate the response to macroautophagy inhibition. Moreover, both cell types manifested synergistic growth inhibition when treated with chloroquine plus the tyrosine kinase inhibitors erlotinib or sunitinib, suggesting that the antiproliferative effects of chloroquine are independent of its suppressive actions on autophagy.

KRAS Cold Turkey: Using microRNAs to target KRAS-addicted cancer.

Human cancers are driven by genetic mutations which cause aberrant activation of pro-growth pathways. Although cancers are uniquely dependent on the pro-growth signaling from oncogenic pathways, efforts to directly target these have been largely unsuccessful. One of the most common and drug resistant oncogenic drivers in colon cancer is the GTPase KRAS. It has been shown that colon cancers with KRAS driver mutations are also 'addicted' to proteins outside of the KRAS pathway due to aberrant re-wiring of cell signaling. A number of genes with a synthetic lethal relationship to mutant KRAS have been previously identified by RNAi screens. MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression, and their expression is frequently dysregulated in cancers. Recently, we have used an innovative functional miRNA screening approach to identify miRNAs that inhibit the survival of KRAS-mutant cells but not KRAS-wild-type cells. MiR-126 was one of the miRNAs that displayed this selective effect. We found that miR-126 induced synthetic lethality in KRAS-Mutant cells via the down-regulation of the polo-like kinase signaling network and a number of genes specifically necessary for the growth of KRAS-Mutant tumors. This study offers a new way forward for exploiting the regulatory power of miRNAs to specifically target aberrant cell signaling in cancer.

RAF Suppression Synergizes with MEK Inhibition in KRAS Mutant Cancer Cells

KRAS is the most frequently mutated oncogene in human cancer, yet no therapies are available to treat KRAS mutant cancers. We used two independent reverse genetic approaches to identify components of the RAS-signaling pathways required for growth of KRAS mutant tumors. Small interfering RNA (siRNA) screening of 37 KRAS mutant colorectal cancer cell lines showed that RAF1 suppression was synthetic lethal with MEK inhibition. An unbiased kinome short hairpin RNA (shRNA)-based screen confirmed this synthetic lethal interaction in colorectal as well as in lung cancer cells bearing KRAS mutations. Compounds targeting RAF kinases can reverse resistance to the MEK inhibitor selumetinib. MEK inhibition induces RAS activation and BRAF-RAF1 dimerization and sustains MEK-ERK signaling, which is responsible for intrinsic resistance to selumetinib. Prolonged dual blockade of RAF and MEK leads to persistent ERK suppression and efficiently induces apoptosis. Our data underlie the relevance of developing combinatorial regimens of drugs targeting the RAF-MEK pathway in KRAS mutant tumors.

Abstract PR03: Targeting CREB-CBP transcription factor complex using small molecule inhibitors with multifunctional anticancer mechanisms.

Abstract Lung cancer is the most common form of cancer in the world and the leading cause of cancer-related deaths in both men and women in the United States. It is estimated that 159,480 lung cancer patients will die in United States in 2013. Approximately 25% of lung adenocarcinomas harbor oncogenic KRAS mutations, and this poses a significant therapeutic challenge, as KRAS mutations are generally associated with poor prognosis and resistance to chemotherapy. There is an unmet therapeutic need for patients with KRAS mutated tumors. Direct pharmacologic targeting of activated KRAS mutant protein has been unsuccessful so far, therefore, other approaches to abrogate KRAS signaling other than through direct inhibition, are being considered. One option to inhibit growth of KRAS mutant tumors may be to target transcription factors, which are often the final regulator of multiple signaling processes, and could potentially be targeted regardless of alterations of upstream signaling components involved in cancer development, progression and invasion/metastasis. CREB is a transcription factors that binds to the specific DNA sequence 5'-TGACGTCA-3', which is known as CRE, in the transcription-regulatory regions of many cAMP-regulated genes with cell-type specificity. Studies have shown that CREB is activated via diverse signaling pathways, including Ras-ERK1/2, PI-3K-Akt, and mTOR signaling pathway. Numerous kinases in these pathways are known to activate CREB in response to a variety of extracellular stimuli (i.e., growth factors, stress signals) and intracellular oncogenic activation mutations. Virtually, almost all of the signaling pathways playing critical roles in carcinogenesis/tumorigenesis are shown to activate CREB. Upon activation, CREB recruit transcriptional coactivator CREBBP (CBP) and directly regulates multiple genes involved in cancer pathogenesis. We previously demonstrated that CREB is a critical transcription factor lung cancer development through its involvement in cell survival, cell cycle progression, proliferation and apoptosis regulation. We, then, first proposed that CREB could be a molecular target for the prevention and treatment of NSCLC. We demonstrated that blocking CREB activity or expression in cancer cells resulted in the suppression of growth and proliferation and induction of cell death. We also reported that CREB regulates expression of pro-inflammatory, pro-angiogenic, and pro-metastatic CXC type chemokine genes and that an inhibitor of CREB blocked the expression and pro-tumorigenic activity of these chemokines. Furthermore, we showed that CREB is highly upregulated and hyperphosphorylated in most of the NSCLC tumor specimens and that this upregulation is significantly associated with poor survival rates as determined in more than 300 cases of NSCLC patients. Thus, these studies lead to the rationale for identification of small molecule inhibitors targeting CREB transcriptional activity described in this study. Here, we will discuss our recent findings that novel small-molecule inhibitors (SMI) targeting the CREB-CBP transcription factors complex are effective in suppression of growth and proliferation of several lung cancer cell lines with various oncogenic mutations, such as KRas-G12D, -G12C, -G12V, or -G12S, and even EGFR-T790M. Furthermore, the small molecule regressed tumors in the KrasG12D/+;p53-/- preclinical mouse lung cancer model. Interestingly, the SMIs also showed similar growth suppressive effect on some pancreatic cancer cell lines and breast cancer cell lines. We found that the SMIs have multifunctional anti-cancer effects including cell cycle arrest, suppression of tumor-promoting autophagy, induction of ER stress, and ultimately cell death. Our results strongly suggest that these novel molecules targeting CREB-CBP transcription factor complex may have potential therapeutic benefit, particularly, cancers harboring various oncogenic mutations, including KRAS and EGFR mutations. This abstract is also presented as Poster B04. Citation Format: Jong Woo Lee, Hee Sun Park, Sin-aye Park, Roy S. Herbst, Jaseok Peter Koo. Targeting CREB-CBP transcription factor complex using small molecule inhibitors with multifunctional anticancer mechanisms. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr PR03.

PIK3CA Mutation Uncouples Tumor Growth and Cyclin D1 Regulation from MEK/ERK and Mutant KRAS Signaling

Mutational activation of KRAS is a common event in human tumors. Identification of the key signaling pathways downstream of mutant KRAS is essential for our understanding of how to pharmacologically target these cancers in patients. We show that PD0325901, a small-molecule MEK inhibitor, decreases MEK/ERK pathway signaling and destabilizes cyclin D1, resulting in significant anticancer activity in a subset of KRAS mutant tumors in vitro and in vivo. Mutational activation of PIK3CA, which commonly co-occurs with KRAS mutation, provides resistance to MEK inhibition through reactivation of AKT signaling. Genetic ablation of the mutant PIK3CA allele in MEK inhibitor-resistant cells restores MEK pathway sensitivity, and re-expression of mutant PIK3CA reinstates the resistance, highlighting the importance of this mutation in resistance to therapy in human cancers. In KRAS mutant tumors, PIK3CA mutation restores cyclin D1 expression and G(1)-S cell cycle progression so that they are no longer dependent on KRAS and MEK/ERK signaling. Furthermore, the growth of KRAS mutant tumors with coexistent PIK3CA mutations in vivo is profoundly inhibited with combined pharmacologic inhibition of MEK and AKT. These data suggest that tumors with both KRAS and phosphoinositide 3-kinase mutations are unlikely to respond to the inhibition of the MEK pathway alone but will require effective inhibition of both MEK and phosphoinositide 3-kinase/AKT pathway signaling.