TL-895, a Highly Selective, Covalent Inhibitor of Bruton's Tyrosine Kinase (BTK), Sensitizes Myeloproliferative Neoplasm (MPN)-Blast Phase Stem Cells to Navtemadlin By Targeting Intrinsic Dysregulated MDM2/p53 and NF-Κb Pathways and Disrupting the Protective Tumor Microenvironment (TME)

We recently reported that navtemadlin (nvtm), a potent, orally available, selective inhibitor of MDM2 (MDM2i), can deplete/eliminate TP53^WT MPN-blast phase (BP) stem cells (SC) in an MPN-BP patient (pt)-derived xenograft (PDX) model (Wang EHA 2023). Single agent nvtm has demonstrated significant clinical activity in pts with advanced myelofibrosis (MF) (Vachhani EHA 2023, Verstovsek Future Oncology 2022). We hypothesized that effects of nvtm on MPN-BP, which is characterized by multiple SC subclones, could be enhanced by combination with agents that target additional MPN-BP SC vulnerabilities, such as hyperactivation of p65 subunit of NF-κB (Fisher Leukemia 2017; our unpublished data) and dependence on TME support.

BTK and bone marrow (BM) kinase X-linked (BMX) are key Tec family members expressed in cells of myeloid origin. Here, we show BTK is constitutively activated in MPN-BP SC subpopulations relative to normal donor (ND) hematopoietic SCs (HSC) and progenitors [phospho (p)-BTK _Y233 mean fluorescence intensity fold change (FC)]: CD34 ⁺CD38 ^- cells: 1.34±0.18, HSC/multipotent progenitor (MPP): 1.50±0.23, multi-lymphoid progenitor (MLP): 1.59±0.59, megakaryocyte-erythroid progenitor (MEP): 1.56±0.49; p <0.05]. TL-895 is a highly selective, potent, orally available, second-generation covalent inhibitor of BTK and BMX, currently under clinical evaluation for treatment of pts with CLL (NCT02825836) and MF (NCT04655118). We used multi-modal single-cell analysis to study mechanistic effects of combination TL-895 + nvtm on dysregulated intracellular signaling pathways in primary MPN-BP SC, and investigated the combination in a PDX model of MPN-BP.

In vitro treatment with TL-895 alone (100 nM-5 µM) did not affect growth of ND CD34 ⁺ cells [FC (TL-895 vs. vehicle, veh): 2.5 µM: 1.27±0.11, 5 µM: 0.97±0.14, n=3)] but modestly decreased growth of primary TP53^WT MPN-BP CD34 ⁺ cells, in a dose dependent fashion [FC (TL-895 vs. veh): 2.5 µM: 0.88±0.11, 5 µM: 0.62±0.12, n=4. p<0.05). Addition of TL-895 (5 µM) to nvtm (400 nM) resulted in much greater reductions in MPN-BP CD34 ⁺ and CD34 ⁺CD38 ^- cell numbers, along with increased apoptosis (n=6, p<0.05, TL-895 + nvtm vs. veh, TL-895, or nvtm alone). By contrast, at these doses, combination TL-895 + nvtm did not affect ND HSC subpopulations (n=3). We previously demonstrated that, multiple MPN-BP SC subclones contribute to the development of MPN-BP irrespective of clone size, and minor subclones may be responsible for clonal evolution and disease progression in most cases (Wang JCI insight 2022). Strikingly, single cell mutational profiling and epitope analysis of primary MPN-BP CD34 ⁺ cells treated with TL-895 + nvtm showed depletion of all 8 distinct MPN-BP SC subclones with differing variant allele frequencies, each containing 2-3 oncogenic mutations in WT1, ETV6 and/or KRAS. Combination TL-895 + nvtm treatment depleted 7 of 8 subclones to a greater degree than either agent alone (Fig. 1). Bulk RNA-seq and gene set enrichment analysis showed that combination TL-895 + nvtm synergistically upregulated p53 pathway gene transcription ( CDKN1 HDM2, DDB2, BTG2, GADD45A, FAS, FUCA1, S100A10, ATF3) in primary MPN-BP CD34 ⁺ cells. Mass cytometry analysis and intracellular flow cytometry of MPN-BP CD34 ⁺ cells treated with combination TL-895 + nvtm further revealed significant (p<0.05) upregulation of p53 and BAX proteins and decreases in the anti-apoptotic factors BCL-xL and activated NF-κB (p-p65), as well as significant reductions in DNA synthesis and protein markers associated with glycolysis (p-AMPK, MCT1). Downregulation of NF-κB activation might result from decreases in the upstream modulators p-S6 and p-4EBP1 (p<0.05, TL-895 + nvtm vs. veh). Treatment in vivo withTL-895 promoted dose-dependent egress of hCD45 ⁺CD34 ⁺ cells from BM into the spleen and peripheral blood (PB) of MPN-BP PDX mice (Fig. 2). Furthermore, a limited course of combination TL-895 + nvtm treatment for 3 cycles led to clearance of hCD45 ⁺CD34 ⁺ cells from the BM, spleen and PB of MPN-BP PDX mice (Fig. 2); neither treatment had this effect when administered alone. Our results indicate that BTKi therapy might increase susceptibility of MPN-BP SC to MDM2i therapy, by upregulating p53 activity and dampening NF-κB signaling, and also by disrupting protective TME interactions that sustain MPN-BP SC. This novel combination merits further clinical investigation in advanced phase MPN.