Suppression of CML Progenitor but Not Stem Cells Requires Simultaneous Inhibition of KIT and BCR-ABL1.
Authors: Gu Zhimin , Corbin Amie S. , O'Hare Thomas , Eiring Anna M. , Zhang Tian yi , Druker Brian J. , Deininger Michael W. 
Published: October 2019

Abstract
Abstract 2778
In chronic myeloid leukemia (CML), imatinib and other tyrosine kinase inhibitors (TKIs) inhibit BCR-ABL1 tyrosine kinase activity but also target additional kinases including KIT. The role of KIT inhibition in the therapeutic efficacy of TKIs is controversial. We used TKIs with selective activity against ABL (PPY-A) or KIT (BAW667) and genetic tools to assess the role of KIT signaling for growth of CML cell lines and primary CML progenitor and stem cells. In Mo7eBCR-ABL1 or newly diagnosed CML CD34+ progenitor cells, immunoblotting confirmed that PPY-A (1 μM) suppresses BCR-ABL1 phosphorylation but not KIT tyrosine phosphorylation. In contrast, treatment of cells with a KIT-blocking antibody (K44.2, 200ng/mL), shRNA targeting KIT (shKIT), or the KIT selective inhibitor BAW667 (1 μM), suppressed KIT activity without affecting BCR-ABL1 kinase activity. Therefore, these systems are suitable to isolate the role of BCR-ABL1 vs. KIT inhibition.
Treatment of Mo7eBCR-ABL1 cells with PPY-A resulted in suppression of growth by 91.7% (p<0.003). When PPY-A was combined with KIT activation by SCF, proliferation was restored, indicating KIT signaling must be inactivated to induce cell death by BCR-ABL1 inhibition. Immunoblot analysis of Mo7eBCR-ABL1 cells revealed that culture in SCF rapidly activated AKT and ERK1/2 in the presence but not absence of PPY-A. Simultaneous inhibition of AKT with LY294002 abolished SCF-mediated rescue of cell proliferation, whereas ERK1/2 inhibition with PD98059 only partially abrogated SCF rescue. These data indicate that SCF rescue of Mo7eBCR-ABL1 cells upon BCR-ABL1 inhibition critically depends on AKT.
To assess BCR-ABL1 vs. KIT inhibition in primary cells, CD34+ cells from newly diagnosed CML patients (n=4) and normal controls (n=3) were cultured in semisolid medium supplied with IL-3 and GM-CSF (no SCF), in the presence of 1 μM PPY-A combined with shKIT or 1 μM BAW667. KIT inhibition by shKIT or 1 μM BAW667 reduced CFU-GM formation by 40% compared to controls (p<0.04) even in the absence of SCF, with no effects were seen in normal CD34+ cells, indicating that BCR-ABL1-dependent KIT activation occurs in the absence of SCF stimulation. PPY-A reduced colony formation by 54.7%, while PPY-A plus shKIT and PPY-A plus BAW667 suppressed CFU-GM colony formation by 79.7% and 72.1%, comparable to the effects of imatinib (71.9%). Addition of SCF partially rescued colony growth from the effects of PPY-A, consistent with results on Mo7eBCR-ABL1 cells. In a separate set of experiments lineage-negative (Lin−) cells from newly diagnosed patients (n=4) were cultured on HS-5 stromal cells containing K44.2, PPY-A, K44.2 plus PPY-A or 2 mM imatinib, followed by clonogenic assays. Only the PPY-A / K44.2 combination suppressed CFU-GM; isolated BCR-ABL1 or KIT block did not. These data demonstrate that both BCR-ABL1 and KIT contribute to CML progenitor cell survival under physiologically relevant conditions, and that inhibition of both pathways is required for imatinib-mediated suppression of CML progenitor cells.
To assess the role of KIT vs. BCR-ABL1 inhibition on primitive CML cells we performed long-term culture-initiating cell (LTC-IC) assays on M2–10B4 murine stromal cells, using Lin− cells from newly diagnosed patients (n=3). Cultures were performed with K44.2, PPY-A, K44.2 plus PPY-A or 2 mM imatinib, with colonies plated at 1, 3, and 6 weeks. At 1 week colonies were reduced by 30% with K44.2 and 70% with PPY-A, but by 90% with the PPY-A / K44.2 combination or with imatinib. In contrast, at 6 weeks colony formation was unaffected by K44.2 but reduced by >95% with PPY-A, the PPY-A / K44.2 combination or imatinib. Week 3 colony growth was intermediate. Consistent with the LTC-IC assay, KIT inhibition with BAW667 enhanced PPY-A suppression of colony formation in Lin−CD34+CD38+ progenitor cells from newly diagnosed patients (n=3) by 18.7% (p<0.05), with no significant effect on primitive Lin−CD34+CD38− cells (7.7%, p=ns).
Our findings suggest KIT inhibition is much more critical for suppression of mature progenitors compared to primitive CML cells. Since AKT is active in CML progenitors but suppressed by TGFβ in stem cells (Nature, 2010;463(7281):676; JCI, 2011;121(1):396), we speculate that upon BCR-ABL1 inhibition CML progenitors but not stem cells switch to an SCF-dependent mode of AKT activation, which renders these cells uniquely sensitive to dual inhibition of BCR-ABL1 and KIT signaling.

Disclosures:
No relevant conflicts of interest to declare.