Intermittent dosing of KO-2806 leads to tumor regressions in combination with RAS inhibitors.

Abstract Antiangiogenic tyrosine kinase inhibitors (TKI) targeting vascular endothelial growth factor receptor (VEGFR) remain the backbone of therapy in advanced renal cell carcinoma (RCC). However, durability of responses is limited and resistance typically arises. Thus, there is an urgent need for therapeutic agents that enhance responses to TKIs, including in patients who progress on prior TKI therapies. In this study, we show that the farnesyl transferase inhibitor (FTI) KO-2806 inhibits mammalian target of rapamycin complex 1 (mTORC1) signaling in endothelial cells to enhance the antiangiogenic properties of TKIs. This translates to tumor regressions and robust inhibition of tumor neovascularization in preclinical models of RCC exposed to the combination of KO-2806 and anti-VEGFR TKIs. KO-2806 also sensitizes tumors previously progressing on anti-VEGFR TKIs, suggesting potential benefits of KO-2806 as a combination partner across the treatment continuum in RCC.

The combination of KO-2806 and mutant-selective KRAS or pan-RAS inhibitors inhibits mTOR signaling in CRC models and is tolerable in vivo

Menin scaffolds the oncogenic histone-lysine-N-methyltransferase (KMT2A)-fusion protein (FP) complex in KMT2A-r and wild-type KMT2A complex in NPM1-m acute myeloid leukemia (AML). Menin inhibitors (MIs) are effective in KMT2A-r AML and NPM1-m AML. However, not all patients respond to MIs as monotherapy. In this preclinical study, we demonstrate that the MI ziftomenib, in combination with the XPO1 inhibitor selinexor, synergistically inhibited the growth of multiple KMT2A-r and NPM1-m AML cell lines (CI<1). The combination suppressed colony formation in primary CD34+ KMT2A-r progenitor cells without affecting normal stem cells. Robust apoptosis and decreased G2/M populations were also evident. The combination downregulated HOXA9 and MEIS1 while upregulating monocytic differentiation marker CD11b in both the AML molecular signatures. RNA sequencing and proteomic analysis in KMT2A-r revealed suppression of multiple bona fide menin-KMT2A target genes. Our mechanistic studies also identified a novel role of XPO1 in stabilizing menin's binding to chromatin and its interactions with KMT2A and KMT2A/MLLT3. XPO1 inhibitor-mediated disruption of these interactions, particularly in combination with ziftomenib, synergistically impairs oncogenic transcriptional programs. In vivo, combination therapy improved survival in both MV4;11 and OCI-AML3 cell line and primary patient-derived KMT2A-r and NPM1-m AML xenograft models in NSG mice, effective even at reduced drug doses. These preclinical findings demonstrate that simultaneous inhibition of the menin-KMT2A interaction and XPO1 can be a more effective translational strategy for treating KMT2A-r and NPM1-m AML than MI monotherapy to deepen responses and delay/prevent relapses.

KO-2806 addition re-sensitizes tumors to KRAS inhibition

Pharmacokinetic profile, tolerability, and antitumor efficacy of KO-2806.

Antitumor efficacy and tolerability of combined KO-2806 and MRTX1133 or RMC-6236 in KRAS-mutant CRC xenograft models.

Resistance remains a key issue limiting the clinical benefit from RAS-targeting therapeutic agents and necessitates combination approaches. In this study, we identified persistent mTORC1 activity in preclinical KRAS-mutant non-small cell lung cancer (NSCLC) and colorectal cancer models as a frequent, nongenetic driver of inherent and adaptive resistance to RAS inhibition. This vulnerability was targetable with the farnesyl transferase inhibitor darlifarnib (KO-2806), which blocks mTORC1 activation via RHEB while sparing mTORC2 to limit associated toxicities. The addition of KO-2806 to NSCLC or colorectal cancer tumors progressing on mutant-selective RAS inhibitors led to rapid and durable tumor regression. In contrast, switching from mutant-selective to pan-RAS inhibitor monotherapy resulted in only stasis of NSCLC tumors and had no effect on colorectal cancer tumor progression. Furthermore, the addition of KO-2806 rescued sensitivity of progressing tumors to the pan-RAS inhibitor RMC-6236. These results establish mTORC1 as an important mediator of escape from RAS inhibition and highlight KO-2806 as a promising RAS companion inhibitor in patients with prior RAS inhibitor exposure.Significance: KO-2806 salvages RAS inhibitor activity by controlling parallel mTORC1 in RAS inhibitor-resistant tumors in which vertical inhibition of MAPK is insufficient to restore sensitivity, providing a combination strategy for resistant patients.

Anti-tumor efficacy and tolerability of combined KO-2806 and cabozantinib in RCC xenograft models.

KO-2806 is tolerable in combination with anti-VEGFR TKIs in RCC tumors progressing on TKIs.

Instructions for synthesis of KO-2806

(A) Immunoblots confirming MEF2C overexpression in MOLT4 cells. (B) Eight-day growth curves for control vs MEF2C OE MOLT4 cells. (C) Co-treatment with ziftomenib (0.3 µM) and CDK1/2 inhibitor (0.3 µM) in control vs MEF2C OE MOLT4 cells.

Antitumor efficacy and tolerability of combined adagrasib and KO-2806 in KRASG12C-mutant NSCLC xenograft models.

KO-2806 does not inhibit HUVEC tube formation in vitro.

KO-2806 induces cell cycle arrest in RCC tumors.

Tolerability of prolonged treatment with KO-2806/RAS inhibitor combinations in mice

Identifying ideal candidates for cancer therapies is challenging, especially with multiple oncogenic variants involved. In head and neck squamous cell carcinoma, the PI3Kα-AKT-mTOR and HRAS-MAPK pathways are frequently dysregulated. This study demonstrates how a quantitative systems pharmacology (QSP) model can help optimize clinical trial design by guiding patient selection and dosing strategies based on oncogenic genotypes. A QSP model was developed to capture the experimentally observed dynamics of the HRAS and PI3K pathways across five molecularly defined patient cohorts in the KURRENT-HN Phase I/II trial (NCT04997902). The model assessed which genotypes would benefit the most from combination therapy with tipifarnib (farnesyl transferase inhibitor) and alpelisib (PIK3CA inhibitor). Simulation results identified the PIK3CA gain of function (GOF) as the genotype most likely to benefit. Virtual population analysis of PIK3CA GOF with dose escalation to 600 mg b.i.d. tipifarnib and 250 mg q.d. alpelisib suggested that a higher tipifarnib dose could enhance tumor response, potentially due to significant dependency of the PIK3CA-mutant cells on mTORC1 signaling. These simulations were consistent with the clinical data. This key dependency can be targeted by tipifarnib by blocking farnsylation of RHEB, an essential activator of mTORC1. Vpop responders showed that reduced intracellular mTOR activity in simulations increased the likelihood of tumor volume reduction. Global sensitivity analysis identified compensatory feedback, tumor proliferation rate, and PI3K-mTOR crosstalk as key determinants of tumor response. This novel QSP application exemplifies an innovative bottom-up modeling approach to support patient selection and dosing strategies for future clinical studies.

Tolerability of KO-2806 with or without axitinib, cabozantinib, or lenvatinib in Caki1 RCC xenograft tumor model.

Combination of KO-2806 and adagrasib inhibits mTOR signaling and induces tumor regression in KRASG12C-mutant NSCLC models