KRAS Wild-type Lines Research Articles

Abstract C018: Primary tumor data analysis reveals novel synthetic lethal dependencies between KRAS mutation and the spliceosome

Abstract Identification of mutation-specific targeted therapies is a critical challenge in precision medicine. Synthetic lethality provides the basis for an approach to identify new therapeutic targets for mutations that are difficult to target directly, since in synthetic lethal (SL) interactions, an alteration in one gene leads to dependency on a second gene. Neither alteration by itself is essential for survival, but together these alterations lead to cancer cell death. We previously developed a new computational method, Mining Synthetic Lethals (MiSL), that can infer relationships from primary human tumor genomic and transcriptomic data and enable the identification of SL interactions in the context of native human tumors. The underlying assumption of MiSL is that SL partners of a mutation will be selectively amplified or never deleted in primary tumor samples harboring the mutation. Here, we used MiSL to identify novel SL partners of KRAS mutations in lung cancer. KRAS activating mutations are highly prevalent in lung cancer and aside from early clinical data on a novel covalent inhibitor for KRAS G12C mutations, there is no reported treatment to date for patients with KRAS mutations. We applied MiSL to identify SL interactions with KRAS G12 mutations in non-small cell lung cancer (NSCLC) and identified 220 SL partner genes. Some of these genes were foreseeable dependencies, such as MAPK, growth factor and inflammatory signaling associated genes; while others were novel SL candidates and included genes associated with splicing. Since we have access to a splicing-modulating drug in preclinical development, sudemycin D6 (SD6) which targets SF3B1, a spliceosome subunit, we focused on testing the SF3B1 interactors identified by MiSL, SLU7 and SRRM2. To enable validation studies, we first verified KRAS growth dependency in a panel of NSCLC cell lines using siRNA knock-down (KD). Next, we tested siRNA KD of predicted MiSL candidates on growth of KRAS-dependent (H358 and H441 – KRAS mutant) and KRAS-independent (H2228 – KRAS WT) cell lines. We confirmed that the KD of SLU7 expression reduced viability of KRAS mutant (75 to 85% reduced) but not KRAS wild-type (WT) cells lines. Similar differential viability was also observed with siRNA KD of SF3B1. Pharmacological inhibition of SF3B1 using SD6 also demonstrated that KRAS mutant cell lines were more sensitive to SD6 compared to a KRAS WT line (>5-30x). Finally, we validated these findings in an in vivo tumor xenograft model by treating KRAS WT and KRAS mutant tumors with SD6 and showed that the KRAS mutant tumors were significantly more inhibited by SD6 than KRAS WT tumors. Studies are ongoing to further confirm the SL interaction in lung cancer cell lines that are isogenic except for the KRAS mutation. In conclusion, SL predictions based on primary KRAS G12 mutant tumors indicated that splicing modulation is a vulnerability of KRAS tumors. Finally, synthetic lethal targets identified by primary tumor data mining can provide new therapeutic targets for KRAS specific mutant cancers, which can lead to personalized treatment options for NSCLC cancer patients. Citation Format: Claire E Repellin, Puja Patel, Yihui Shi, Helena Gong, Leena Shewade, Thomas Webb, Lidia Sambucetti, Subarna Sinha. Primary tumor data analysis reveals novel synthetic lethal dependencies between KRAS mutation and the spliceosome [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C018. doi:10.1158/1535-7163.TARG-19-C018

Abstract 2954: TWIST1 is a critical mediator of KRAS mutant tumorigenesis in human non-small cell lung cancer

Abstract A large fraction of non-small cell lung cancers (NSCLC) have mutant KRAS, which is associated with poor response to current cytotoxic therapy and a poor prognosis. Although the KRAS signaling pathway has been well characterized, no current therapies target this critical oncogene. Several studies have demonstrated that bypass of senescence in Kras-mediated adenocarcinoma mouse models is essential for tumorigenesis. Therefore, activation of senescence in KRAS mutant NSCLC may be an effective therapeutic strategy. We recently demonstrated that the basic helix loop helix transcription factor Twist1 cooperates with mutant Kras to induce lung adenocarcinoma in transgenic mouse models and that inhibition of Twist1 in these models led to activation of Kras-induced senescence and tumor stasis. In the current study, we examine the role of TWIST1 in KRAS mutant human NSCLC. Silencing of TWIST1 in multiple KRAS mutant NSCLC cell lines resulted in dramatic growth inhibition and either reactivation of oncogene-induced senescence or in some cases, apoptosis. Similar effects were also observed in four KRAS wild type lines, including cell lines with key driver mutations including a cell line with an activating EGFR mutation and a cell line with c-Met amplification. Gene set enrichment analysis of NSCLC cell lines after silencing of TWIST1 revealed a striking cell cycle arrest gene signature. Growth inhibition by silencing of TWIST1 was independent of p53 or Rb/p16 mutational status. Furthermore, activation of oncogene-induced senescence by TWIST1 silencing did not require previously defined mediators of senescence, p21 and p27, nor could this phenotype be rescued by overexpression of SKP2. To extend these observations in vivo, TWIST1 was silenced in both KRAS mutant and wildtype cell lines and these cells were implanted in NOD-SCID mice to assess tumor formation. Interestingly, silencing of TWIST1 in xenograft models preferentially inhibited KRAS mutant tumor formation suggesting that TWIST1 plays a critical in mediating KRAS tumorigenesis. Finally, inducible silencing of TWIST1 resulted in significant growth inhibition of established xenograft KRAS mutant tumors. Together these findings suggest TWIST1 is essential for the establishment and maintenance of KRAS mutant NSCLC tumors and silencing of TWIST1 in KRAS mutant NSCLC represents a novel and promising therapeutic strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2954. doi:1538-7445.AM2012-2954

Abstract C43: Dasatinib resensitizes Kras mutant colorectal tumors to cetuximab

Abstract Several recent clinical trials have demonstrated a strong correlation between Kras mutational status and resistance to cetuximab in metastatic colorectal cancer (mCRC). The work presented here sought to further understand the resistance of Kras mutant tumors to cetuximab and analyze dasatinib, a potent FDA approved SRC family kinase inhibitor, and its ability to overcome cetuximab resistance in this genetic setting. We analyzed 16 CRC lines for cetuximab growth response in vivo, expression of EGFR, SRC, and Kras mutational status. From this analysis we selected three Kras mutant lines (HCT116, LS180, LoVo), one Kras wild type line (SW48), and one EGFR negative line (Colo320DM) for further studies. Approximately two million cells from each line were inoculated in the dorsal flanks of athymic nude mice and allowed to grow to 100mm cubed. At that time mice were given 0.3mg of cetuximab twice weekly for 2 to 4 weeks. All Kras mutant lines were resistant to cetuximab in vivo, whereas the Kras wild type line showed significant tumor growth inhibition to cetuximab therapy. Each Kras mutant line was then analyzed for response to dasatinib alone. Mice were given 30mg/kg of dasatinib fives times weekly by oral gavage for 2 to 4 weeks. Results of these experiments indicated that Kras mutant tumors exhibited very little response to dasatinib monotherapy. However, dasatinib given in combination with cetuximab lead to significant tumor growth delay relative to vehicle controls. Collectively, these data suggest that Src and its family of kinases cooperate with the EGFR in the Kras mutant, mCRC setting and dual blockade has a distinct anti-tumor effect. These studies strengthen the rationale for the design of clinical trials combining dasatinib and cetuximab in the Kras mutant mCRC setting. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C43.