Lian-Sheng Li

The KRASG12C mutation is a critical therapeutic target in the management of solid tumors, owing to its role in oncogenic signaling. Recent advances in covalent inhibitors that target mutant KRAS cysteine-12 have demonstrated the potential to halt aberrant signaling associated with this historically “undruggable” target. Here, we report the identification of 6-cyanoquinazoline covalent irreversible KRASG12C inhibitors. Lead optimization used structure-based design to identify novel switch-II pocket-binding motifs and in silico models to forecast in vitro metabolic stability and permeability. Human dose was improved by maximizing the rate of covalent modification (kobs/[I]) of KRASG12C-GDP, along with optimizing ADME parameters, to identify potent, orally bioavailable lead molecule 13de which demonstrated significant antitumor efficacy in the NCI-H1373 human lung adenocarcinoma xenograft model. Studies evaluating KRASG12C-GDP covalent target engagement, pharmacokinetics, and tumor growth inhibition estimated the efficacious human dose of 13de to be 192 mg administered once daily (QD), using allometric scaling.

The protein-protein interaction between menin and KMT2A (histone lysine methyltransferase 2A) plays a critical role in acute leukemia with KMT2A rearrangements, nucleophosmin 1 (NPM1) mutations and nucleoporin 98 rearrangements, and represents an emerging opportunity for therapeutic intervention. Here, we report development and comprehensive evaluation of the activity of ziftomenib as an orally bioavailable, highly potent and selective small molecule inhibitor of the menin-KMT2A interaction. In leukemia cells and primary patient samples with the menin-KMT2A dependency, ziftomenib profoundly inhibited proliferation, reduced clonogenic potential and induced differentiation, which was associated with strong downregulation of the menin-KMT2A target genes, including MEIS1, HOXA9 and HOXB2. In xenografts and patient-derived xenograft models of KMT2A-rearranged leukemia, ziftomenib induced leukemia regression or reduced leukemia burden, accompanied by a pronounced reduction of the menin-KMT2A target genes. We next assessed ziftomenib against four MEN1 (gene encoding menin) mutants (T349M, M327I, G331R, G331D) associated with clinical resistance to another menin inhibitor revumenib. Ziftomenib retained anti-leukemic activity against T349M mutant cells and demonstrated low-nanomolar potency (GI50≤25 nM) against G331R cells, despite several-fold reduced potency relative to MEN1 wild-type cells, whereas M327I and G331D mutants were resistant. The crystal structures of ziftomenib in complex with menin wild-type, T349M or G331R mutants revealed a similar binding mode of ziftomenib to these menin variants, rationalizing potent inhibitory activity towards these mutants. Ziftomenib has recently received FDA approval for adult patients with NPM1-mutated acute myeloid leukemia and continues to be evaluated clinically in leukemias with NPM1 or KMT2A alterations, both as monotherapy and in combinations.

Combined targeting of KRAS-G12C, mTOR, and IGF1R enhances and extends the response to recently developed KRAS-G12C inhibitors in lung cancer models.

Purpose: KRAS mutant lung cancers have been recalcitrant to treatments including those targeting the MAPK pathway. Covalent inhibitors of KRAS p.G12C allele allow for direct and specific inhibition of mutant KRAS in cancer cells. However, as for other targeted therapies, the therapeutic potential of these inhibitors can be impaired by intrinsic resistance mechanisms. Therefore, combination strategies are likely needed to improve efficacy. Experimental Design: To identify strategies to maximally leverage direct KRAS inhibition we defined the response of a panel of NSCLC models bearing the KRAS G12C activating mutation in vitro and in vivo. We used a second-generation KRAS G12C inhibitor, ARS1620 with improved bioavailability over the first generation. We analyzed KRAS downstream effectors signaling to identify mechanisms underlying differential response. To identify candidate combination strategies, we performed a high-throughput drug screening across 112 drugs in combination with ARS1620. We validated the top hits in vitro and in vivo including patient derived xenograft models. Results: Response to direct KRAS G12C inhibition was heterogeneous across models. Adaptive resistance mechanisms involving reactivation of MAPK pathway and failure to induce PI3K-AKT pathway inactivation were identified as likely resistance events. We identified several model-specific effective combinations as well as a broad sensitizing effect of PI3K-AKT-mTOR pathway inhibitors. The G12Ci+PI3Ki combination was effective in vitro and in vivo on models resistant to single agent ARS1620 including patient derived xenografts models. Conclusions: Our findings suggest that signaling adaptation can in some instances limit the efficacy of ARS1620 but combination with PI3K inhibitors can overcome this resistance.

Activating mutations in KRAS are among the most common tumor driver mutations. Until recently, KRAS had been considered undruggable with small molecules; the discovery of the covalent KRAS^G12C inhibitors ARS-853 and ARS-1620 has demonstrated that it is feasible to inhibit KRAS with high potency in cells and animals. Although the biological activity of these inhibitors has been described, the biochemical mechanism of how the compounds achieve potent inhibition remained incompletely understood. We now show that the activity of ARS-853 and ARS-1620 is primarily driven by KRAS-mediated catalysis of the chemical reaction with Cys12 in human KRAS^G12C, while the reversible binding affinity is weak, in the hundreds of micromolar or higher range. The mechanism resolves how an induced, shallow and dynamic pocket not expected to support high-affinity binding of small molecules can nevertheless be targeted with potent inhibitors and may be applicable to other targets conventionally considered undruggable.

KRAS gain-of-function mutations occur in approximately 30% of all human cancers. Despite more than 30 years of KRAS-focused research and development efforts, no targeted therapy has been discovered for cancers with KRAS mutations. Here, we describe ARS-853, a selective, covalent inhibitor of KRAS^G12C that inhibits mutant KRAS–driven signaling by binding to the GDP-bound oncoprotein and preventing activation. Based on the rates of engagement and inhibition observed for ARS-853, along with a mutant-specific mass spectrometry–based assay for assessing KRAS activation status, we show that the nucleotide state of KRAS^G12C is in a state of dynamic flux that can be modulated by upstream signaling factors. These studies provide convincing evidence that the KRAS^G12C mutation generates a “hyperexcitable” rather than a “statically active” state and that targeting the inactive, GDP-bound form is a promising approach for generating novel anti-RAS therapeutics. Significance: A cell-active, mutant-specific, covalent inhibitor of KRAS^G12C is described that targets the GDP-bound, inactive state and prevents subsequent activation. Using this novel compound, we demonstrate that KRAS^G12C oncoprotein rapidly cycles bound nucleotide and responds to upstream signaling inputs to maintain a highly active state. Cancer Discov; 6(3); 316–29. ©2016 AACR. See related commentary by Westover et al., p. 233. This article is highlighted in the In This Issue feature, p. 217