Introduction RAS mutations are prevalent in AML, but efforts to target the mitogen activated protein kinase (MAPK) effector pathway have been largely unsuccessful. Belvarafenib is a type II pan-Raf kinase inhibitor that is active against both monomeric and dimeric mutant B-Raf proteins as well as wild-type (WT) Raf homo- and heterodimers (Yen et al. 2021). We observed promising anti-leukemia activity of belvarafenib as a single agent and in combination with the allosteric MEK inhibitor cobimetinib in AML models harboring oncogenic NRAS/Nras or KRAS/Kras mutations (2023 ASH Annual Meeting, Abstract #4172). Belvarafenib treatment unexpectedly had minimal effects on phosphorylated (p) ERK levels and downregulated mTORC1 signaling in multiple RAS-mutant AML cell lines. In this study, we further analyzed belvarafenib's mechanism of action and identified and analyzed candidate resistance mutations that emerged after in vivo treatment. Methods Mouse AMLs that were generated using retroviral insertional mutagenesis were transplanted and treated as previously described (Li et al. 2011, Burgess et al. 2014, 2017). Whole exome sequencing (WES) was performed on DNA extracted from bone marrow cells isolated at euthanasia from recipients of primary Nras- or Kras-mutant AMLs that were treated with either belvarafenib, cobimetinib, belvarafenib + cobimetinib, or vehicle. OCI-AML3 cells were lentivirally transduced with doxycycline-inducible vectors containing constitutively active MEK-DD, a MEK mutation that confers resistance to allosteric MEK inhibitors (L115P), or candidate MEK resistance mutations and cell viability was determined by CellTiter-Glo. Results To investigate whether MAPK pathway inhibition is essential for the inhibitory activity of belvarafenib in AML cells, we expressed doxycycline-inducible MEK-DD and MEKL115P mutant proteins. NRAS-mutant OCI-AML3 cells expressing MEK-DD were resistant to belvarafenib and sensitive to cobimetinib. As expected, MEKL115Pinduced resistance to cobimetinib, but not belvarafenib. To characterize the effects of belvarafenib treatment on mTORC signaling, we treated OCI-AML3 cells with belvarafenib 100nM, cobimetinib 10nM, this combination, or vehicle for 4 or 24 hours (h). While we observed minimal effect of drug treatment at 4h, belvarafenib decreased pAKT, p4EBP1 and p70S6K levels after 24h. At the later time point, cells treated with both drugs displayed profound and synergistic reductions in the levels of all three mTORC target phospho-proteins. Of five independent primary murine AMLs, AML 63A was the most sensitive to belvarafenib and the belvarafenib/cobimetinib combination (median survival of vehicle group, 7d; belvarafenib group, 23d; combination group, 32d; p = 0.003). WES of DNA extracted from the bone marrows of mice with refractory leukemia uncovered distinct Map2k1 mutations (K57T, E203K, N122D) in three independent recipient mice treated with the belvarafenib/cobimetinib combination at variant allele frequencies (VAFs) of 10-33% and a Braf L542H mutation in a belvarafenib-treated recipient at a VAF of 21%. MAP2K1 K57T and E203K mutations and a BRAF mutation (L505H) corresponding to L542H in mouse have been reported in patients who developed clinical resistance after treatment with first generation Raf kinase inhibitors. To functionally interrogate putative resistance mechanisms, we expressed MEKK57T and MEKN122D in NRAS mutant OCI-AML3 cells and analyzed them in parallel with control cells expressing wild-type MEK, MEK-DD, or MEKL115P. Whereas MEKN122D had no effect on sensitivity to belvarafenib or cobimetinib, MEKK57T conferred partial resistance to cobimetinib and more pronounced resistance to belvarafenib. Conclusions Belvarafenib and cobimetinib synergistically inhibit the growth of RAS-mutant AML cell lines in vitro and of primary mouse Nras- and Kras-mutant leukemias mutations in vivo. Although belvarafenib has minimal effects on pERK levels in KRAS/NRAS-mutant AML cell lines at clinically achievable concentrations, genetic analysis of MEK-DD and MEKL115P mutations confirmed MAPK pathway inhibition as a major mechanism of action. We are characterizing candidate resistance mutations in addition to Mapk21 K57T that emerged during belvarafenib treatment. Altogether, our data support further investigation of belvarafenib monotherapy and rational drug combinations in AML.

Introduction Outcomes for children with acute myeloid leukemia (AML) remain poor, with ~40% dying from refractory leukemia or treatment-related toxicity (Gamis et al. 2014). NRAS, KRAS, and NF1 mutations occur in over 40% of pediatric AMLs (Bolouri et al. 2018), but efforts to therapeutically target the RAS/mitogen activated protein kinase (MAPK) pathway have been largely unsuccessful. Belvarafenib, a novel type II pan-RAF kinase inhibitor that inhibits mutant monomeric BRAF proteins and activated RAF homo- and heterodimers, has shown promising safety/efficacy data in adult solid cancers (Yen et al. 2021). Here, we investigate belvarafenib in preclinical models of AML, both alone and in combination with the allosteric MEK inhibitor, cobimetinib. Methods We used a panel of NRAS (OCI-AML3, HL-60, THP-1) or KRAS (NOMO-1, NB4, and SKM-1) mutant human AML cell lines. Viability was determined by CellTiter-Glo. Synergy was assessed by Bliss Independence and Chou Talalay methods. Transcriptome and proteomic profiling were performed as previously described (Pucciarelli et al. 2020). Mouse AMLs were generated using retroviral insertional mutagenesis (Li et al. 2011). Cryopreserved primary AML cells were injected intravenously into sublethally irradiated recipients that were then treated daily with vehicle, belvarafenib, cobimetinib, or the combination until disease progression. Survival curves were generated using Kaplan-Meier analysis. Results Belvarafenib inhibited the growth of AML cells lines with nanomolar potency at IC50 values ranging from 48nM (OCI-AML3) to 310nM (SKM-1). In all cell lines, belvarafenib and cobimetinib were highly synergistic. Western blotting of OCI-AML3 cells treated with either belvarafenib or cobimetinib at their respective IC50 values (50nM belvarafenib, 20nM cobimetinib) for 4 or 24 hours demonstrated discordant effects on downstream MAPK effector proteins (Fig. 1). Whereas cobimetinib potently suppressed phosphorylated ERK (pERK), belvarafenib unexpectedly had no effect. Phosphorylated S6 (pS6) levels were not reduced by either belvarafenib or cobimetinib at their respective IC50 values, but were dramatically reduced by the drug combination. To further investigate these unanticipated biochemical findings, we treated OCI-AML3 cells with belvarafenib, cobimetinib or the combination and performed kinome profiling and transcriptome sequencing (RNAseq) analysis. Exposure to 10nM cobimetinib inhibited several kinases at 4 hours with rebound activation of kinases involved in cell cycle progression at 24 hours. Cells treated with 100nM belvarafenib displayed down-regulation of multiple kinases that persisted at 24 hours. Cells treated with the combination at these doses exhibited the most potent inhibition of many kinases; this effect recapitulated the effect of high dose (500nM) belvarafenib, but not high dose (50nM) cobimetinib. At the transcriptional level, cells treated with the combination showed far greater suppression of negative regulators of MAPK signaling (e.g., DUSPs, SPRY2/4, SPRED1/2) than either agent alone, suggesting potent inhibition of the MAPK pathway by the combination. Intriguingly, OCI-AML3 cells treated with high-dose belvarafenib or low-dose combination unexpectedly exhibited profound down-regulation of mTORC1-regulated genes on GSEA analysis. We extended these in vitro data by performing preclinical trials in mice transplanted with 5 independent primary Nras- or Kras-mutant AMLs that received belvarafenib (15mg/kg), cobimetinib (2mg/kg), the combination or vehicle by oral gavage (Fig. 2). The combination was well tolerated. Belvarafenib prolonged survival in all 5 trials; in 3 of 5, the addition of low-dose cobimetinib further enhanced survival (median survival: vehicle, 9 days; cobimetinib, 13 days; belvarafenib, 22 days; combination, 32 days; p < 0.0001). Conclusions Belvarafenib showed activity in 6 of 6 Ras mutant human AML cell lines and in 5 of 5 primary Ras mutant murine AMLs. Belvarafenib and cobimetinib displayed synergy in all AML cell lines and in 3 of 5 murine AMLs treated in vivo. Mechanistically, we identified distinct biochemical and transcriptional effects of RAF dimer and MEK inhibition in AML cells. We are characterizing these further and pursuing causes of resistance in primary Nras- and Kras-mutant mouse AMLs that relapsed after an initial response to treatment.

Glucocorticoids (GCs) are the cornerstone of acute lymphoblastic leukemia (ALL) therapy. Although mutations in NR3C1, which encodes the GC receptor (GR), and other genes involved in GC signaling occur at relapse, additional mechanisms of adaptive GC resistance are uncertain. We transplanted and treated 10 primary mouse T-lineage acute lymphoblastic leukemias (T-ALLs) initiated by retroviral insertional mutagenesis with GC dexamethasone (DEX). Multiple distinct relapsed clones from 1 such leukemia (T-ALL 8633) exhibited discrete retroviral integrations that upregulated Jdp2 expression. This leukemia harbored a Kdm6a mutation. In the human T-ALL cell line CCRF-CEM, enforced JDP2 overexpression conferred GC resistance, whereas KDM6A inactivation unexpectedly enhanced GC sensitivity. In the context of KDM6A knockout, JDP2 overexpression induced profound GC resistance, counteracting the sensitization conferred by KDM6A loss. These resistant "double mutant" cells with combined KDM6A loss and JDP2 overexpression exhibited decreased NR3C1 mRNA and GR protein upregulation upon DEX exposure. Analysis of paired samples from 2 patients with KDM6A-mutant T-ALL in a relapsed pediatric ALL cohort revealed a somatic NR3C1 mutation at relapse in 1 patient and a markedly elevated JDP2 expression in the other. Together, these data implicate JDP2 overexpression as a mechanism of adaptive GC resistance in T-ALL, which functionally interacts with KDM6A inactivation.

Microglia are the resident, innate immune cells of the central nervous system (CNS) and are critical in managing CNS injuries and infections. Microglia also maintain CNS homeostasis by influencing neuronal development, viability, and function. However, aberrant microglial activity and phenotypes are associated with CNS pathology, including autism spectrum disorder (ASD). Thus, improving our knowledge of microglial regulation could provide insights into the maintenance of CNS homeostasis as well as the prevention and treatment of ASD. Control of microglial activity is in part overseen by small, lipid-derived molecules known as endogenous cannabinoids (endocannabinoids). Endocannabinoids are one component of the endocannabinoid system (ECS), which also includes the enzymes that metabolize these ligands, in addition to cannabinoid receptor 1 (CB1) and 2 (CB2). Interestingly, increased ECS signaling leads to an anti-inflammatory, neuroprotective phenotype in microglia. Here, we review the literature and propose that ECS signaling represents a largely untapped area for understanding microglial biology and its relationship to ASD, with special attention paid to issues surrounding the use of recreational cannabis (marijuana). We also discuss major questions within the field and suggest directions for future research.