Belvarafenib Inhibits the Growth of RAS-Mutant Acute Myeloid Leukemia Cell Lines in Vitro and Drives Adaptive Resistance In Vivo

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 MEK^L115P mutant proteins. NRAS-mutant OCI-AML3 cells expressing MEK-DD were resistant to belvarafenib and sensitive to cobimetinib. As expected, MEK^L115Pinduced 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 MEK^K57T and MEK^N122D in NRAS mutant OCI-AML3 cells and analyzed them in parallel with control cells expressing wild-type MEK, MEK-DD, or MEK^L115P. Whereas MEK^N122D had no effect on sensitivity to belvarafenib or cobimetinib, MEK^K57T 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 MEK^L115P 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.