BCL2 Splice Isoform Switching Promotes Leukemia Stem Cell Survival and Sensitivity to a Novel Pan BCL2 Inhibitor
Authors: Goff Daniel , Smith Kristen M , Shih Alice Y , Court-Recart Angela , Sadarangani Anil , Geron Ifat , Chuang Ryan , Balaian Larisa , Wei Jun , Kitada Shinichi , Zhai Dayong , Gotlib Jason , Minden Mark D. , Martinelli Giovanni , Schairer Annelie , Leu Heather , Ma Wenxue , Jiang Qingfei , Rusert Jessica , Dao Kim-Hien T , Shazand Kamran , Volar Milica , de Borja Richard , McPherson John D , Hudson Thomas J , Barrett Christian L , Frazer Kelly A , Wentworth Peggy , Jamieson Christina , Morris Sheldon R , Goldstein Lawrence S. B. , Pellechia Maurizio , Reed John C. , Jamieson Catriona 
Published: October 2019

Abstract
Abstract 2735
Leukemia stem cells (LSC) play a crucial role in the development and progression of chronic myeloid leukemia (CML). Although BCR-ABL targeted tyrosine kinase inhibitors (TKI), such as dasatinib, can eradicate the majority of CML cells, they frequently fail to eliminate the dormant, niche-resident LSC that are hypothesized to drive CML relapse. Cumulative evidence from CML cell lines and CD34+ primary patient cells suggests that increased expression of pro-survival BCL2 family members contributes to TKI resistance and CML progression. However there is a relative dearth of data on BCL2 family expression in primary CML LSC and on the role of these proteins in TKI resistance in selective niches.
Full transcriptome RNA sequencing revealed that LSC switch from pro-apoptotic to pro-survival BCL2 family member splice isoform expression during progression from chronic phase to blast crisis CML. Using splice isoform-specific qRT-PCR, we identified overrepresentation of long (pro-survival) compared with short (pro-apoptotic) MCL1, BCLX, and BCL2 isoforms in blast crisis LSC compared with chronic phase and normal progenitors. Following intrahepatic transplantation of blast crisis LSC into neonatal RAG2−/−gc−/− mice, LSC engrafted in the marrow niche were quiescent, were dasatinib resistant and upregulated BCL2 expression. These data led us to speculate that inhibition of BCL2 in dasatinib-resistant LSC may sensitize LSC to TKI therapy. Treatment with a high-potency, novel pan-BCL2 family inhibitor, sabutoclax, in vitro led to a dose-dependent increase in apoptosis along with a decrease in the frequency of leukemic progenitors compared to vehicle treated controls. Normal human cord blood progenitor cells were less sensitive to sabutoclax treatment with IC50 approximately five times higher than that for blast crisis CML cells (210 nM versus 43 nM). Moreover, sabutoclax treatment did not inhibit cord blood colony formation or colony replating in vitro. Treatment of CML LSC-transplanted mice with sabutoclax led to a significant reduction in LSC burden in all hematopoietic organs analyzed. Sabutoclax treatment in vivo also sensitized surviving bone marrow blast crisis LSC to dasatinib treatment ex vivo. Importantly, there was no reduction in normal progenitor engraftment in bone marrow following sabutoclax treatment. These results demonstrate that marrow niche blast crisis CML LSC survival is driven by overexpression of multiple pro-survival BCL2 family isoforms rendering them susceptible to a novel pan, BCL2 antagonist, sabutoclax, at doses that spare normal hematopoietic progenitors. While BCL2 splice isform switching promotes LSC survival and TKI resistance, pan-BCL2 family member inhibition with sabutoclax eliminates LSC and may form the cornerstone of a clinical strategy to avert cancer progression and relapse.

Disclosures:
No relevant conflicts of interest to declare.