Abstract
The BCL2 inhibitor venetoclax (VEN), in combination with hypomethylating agents, is highly effective in inducing remissions in AML. However, most patients eventually relapse due to activation of oncogenic pathways that stabilize MCL1-a major resistance factor to VEN, or through downregulation of pro-apoptotic proteins by mutant TP53. Monocytic AML cells, which express high MCL1 levels, are inherently resistant to VEN, which itself can induce MCL1 levels. Furthermore, under treatment pressure, leukemic myeloblasts can undergo monocytic differentiation, as revealed by CyTOF pseudo-time analysis. Our previous studies have shown that dual inhibition of MCL1 and BCL2 is highly synergistic in AML models resistant to either agent alone. Notably, cells that develop resistance to this combination in vivo upregulate colony stimulating factor 1 (CSF1) (Zhang, Carter et al, Clinical Lymphoma, Myeloma, & Leukemia 2020).
To determine whether AML cells resistant to BH3 mimetics are sensitive to inhibition of the CSF1R-CSF1 signaling axis.
Protein and cytokine levels were assessed by Western blot and/or flow cytometry. BCL2, MCL1, and CSF1R were inhibited with VEN, AZD5991, and the CSF1R antibody SNDX-6352 (Axatilimab), respectively. Cell viability and apoptosis were measured by flow cytometry (Annexin V/7-aminoactinomycin D) and CyTOF in phenotypically defined cell populations. PDX cells developed from a VEN/Decitabine resistant AML patient were treated previously in vivo with VEN/AZD5991, which significantly extended survival. Leukemia cells collected from these treated mice at moribund stage were transplanted into NSG mice, and treated with SNDX-6352, VEN/AZD-5991, or both. The effects on cytokine production, AML blasts, stem cells, and monocytes were evaluated.
MV4-11 cells with acquired resistance to BH3 mimetics targeting BCL2 or MCL1, peculiarly to VEN and VEN plus MCL-1 inhibitor AMG176, exhibited elevated levels of cytokines including CSF1, TGF-1ß, IL-4, and IL-10 compared to parental cells. Plasma from AML patients (n=26) contained significantly higher CSF1 and other cytokines than healthy controls (n=3), with the highest levels observed in patients resistant to VEN-based therapies (n=20) compared to newly diagnosed patients (n=6). CSF1R blocking antibody SNDX-6352 suppressed ERK and AKT signaling and reduced cytokine production in monocytic leukemia cell lines. The combination of SNDX-6352, VEN, and MCL1 inhibitor AZD5991 more effectively eliminated AML blasts and stem/progenitor cells from VEN resistant patients than any single agent or two drug combinations, especially in TP53-mutant samples. In PDX models, SNDX-6352 reduced human cytokine levels in mouse serum and decreased both circulating and bone marrow blasts, stem/progenitor cells, and immunosuppressive MDSC and monocytic leukemia cells. These effects were more pronounced with the combination of SNDX-6352, VEN, and AZD5991.
CSF1, TGF-1ß, IL-4, IL-10, and numerous other cytokines are increased in BH3 mimetic resistant AML cell lines and patient samples. Inhibition of CSF1R targets ERK and AKT signaling and significantly enhances the cytotoxic effects of BH3 mimetics against AML cells, stem/progenitor cells, and monocytes resistant to VEN. Furthermore, blockage of CSF1R-CSF1 axis suppresses multiple cytokines in vitro and in vivo, and markedly improves the therapeutic efficacy of BH3 mimetics in a VEN/AZD5991/Decitabine triple resistant PDX model.
