Preclinical development of a pan Bcl2 inhibitor for cancer stem cell directed therapy

Overexpression of Bcl-2 family genes may fuel CSC survival. Recent RNA sequencing data demonstrate that human CSC express a panoply of antiapoptotic Bcl-2 isoforms in response to extrinsic signals in vivo, indicating that a pan Bcl-2 inhibitor will be required to abrogate CSC survival. Sabutoclax inhibits growth of blast crisis CML CSC engrafted in RAG2-/-c-/- mice with minimal cytotoxicity toward bax-/-bak-/- cells. Because sabutoclax inhibits all six antiapoptotic Bcl-2 family members including Bcl-2, Mcl-1, Bcl-XL, Bfl-1, Bcl-W and Bcl-B proteins, with good chemical, plasma and microsomal stability, we anticipate that it will have clinical utility for targeting apoptosis resistant human CSC in malignancies Significant progress against milestones in the first year was accomplished and we have made early progress on several milestones projected for Year 2. During this 6 month reporting period, sabutoclax was licensed by a biotech company, Oncothyreon. The license was previously held by Coronado Biosciences. Dr. Pellecchia (SBMRI ) continues to provide sabutoclax to Dr. Jamieson for use in cellular and in vivo studies. SBMRI conducted QC analyses (integrity and purity) on samples’ used in preclinical studies and provided comparative analyses of compound produced by the CMO produced by different methods of synthesis. Importantly, the sabutoclax manufacturing process was optimized allowing scale-up of drug. In formulation studies, a method was developed and qualified that separates impurities and degradation compounds from sabutoclax for quantitation of the drug. Additional solubility and stability studies were performed by Oncothyreon to identify an IV formulation that could be used for both nonclinical studies and the clinic. Several pilot PK studies in mice, rats and dogs, planned for Year 2, were also conducted by Oncothyreon. Through whole transcriptome RNA sequencing Dr. Jamieson showed that Bcl-W was up-regulated in CP and BC progenitors compared to normal CB progenitors. Previous qRT-PCR results for Mcl-1 were confirmed, showing that the long isoform was preferentially expressed in BC CML. Results for Bcl-2 and Mcl-1 were also confirmed at the protein level by FACS analysis and immunohistochemistry of bone marrow (BM) from mice engrafted with human CML CD34+ LSC. Sabutoclax treatment ablated BC CML progenitor cells in vivo and in vitro. Colony formation of BC CML (vs normal progenitor cells) was decreased by sabutoclax in a dose dependent manner. When CML cells were co-cultured with stromal cells or in stroma conditioned media, BCL-2 mRNA expression was increased and colony formation was improved. Knockdown of endogenous BCL2 in BC CML cells by shRNA resulted in decreased colony formation. Preliminary results suggest that BM is a protective niche for BC CML CSC and that sabutoclax may target these niche protected cells. In BC CML engrafted mice, dasatinib increased quiescent BC CML cell engraftment in mouse BM measured by FACS for cell cycle markers. Sabutoclax decreased BCL-2 and MCL1 protein expression by immunohistochemistry staining and decreased quiescent BC CML CSC in BM however sabutoclax increased TUNEL staining in BM suggesting that while dasatinib may increase the number of quiescent BC CML CSC, sabutoclax may do the reverse. High doses of sabutoclax administered in combination with dasatinib resulted in a significant decrease in human cell engraftment in BM versus dasatinib alone. Mice serially transplanted with tissues from combination treated mice had increased survival compared to serial transplants of single agent treated tissues. Human CD34+ cells from the BM of combination treated mice had more cells in cycle than CD34+ cells compared to the BM of mice treated with dasatinib alone. The frequency of CD34+BCL2+ and CD34+MCL1+ BC LSC were significantly lower in BM treated with a combination of sabutoclax and dasatinib suggesting that sabutoclax and dasatinib may act synergistically to increase survival of BC CML engrafted mice.

Dormant cancer stem cells (CSC) contribute to therapeutic resistance and relapse in chronic myeloid leukemia (CML) and other recalcitrant malignancies. Cumulative data demonstrate that overexpression of BCL2 family pro-survival splice isoforms fuels quiescent CSC survival in human blast crisis (BC) CML. Whole transcriptome RNA sequencing data, apoptosis PCR array and splice isoform specific qRT-PCR demonstrate that human CSC express anti-apoptotic long BCL2 isoforms in response to extrinsic signals in the marrow niche, indicating that a pan BCL2 inhibitor will be required to abrogate CSC survival. Sabutoclax, a novel pan BCL2 inhibitor, prevents survival of BC CSC engrafted in RAG2-/-c-/- mice, commensurate with downregulation of pro-survival BCL2 splice isoforms and proteins, and sensitizes CSC to a BCR-ABL inhibitor, dasatinib, while exerting minimal cytotoxicity toward normal hematopoietic stem cells. Because sabutoclax inhibits all six anti-apoptotic BCL2 family members, with good chemical, plasma and microsomal stability, in addition to a scaleable production process, we anticipate that it will have broad clinical utility for targeting apoptosis resistant quiescent human CSC in a number of recalcitrant malignancies as featured in our recent lead article (Goff D et al, Cell Stem Cell. 2013 Mar 7;12(3):316-28). Significant progress against milestones in the second year was accomplished and we have made early progress on several milestones projected for Year 3. Whole transcriptome RNA sequencing, qRT-PCR array and splice isoform specific qRT-PCR analysis performed on FACS purified progenitors derived from 8 CP, 8 BC and 6 normal samples demonstrated splice isoform switching favoring pro-survival long isoform expression during progression from CP to blast BC CML and in CSC engrafted in the bone marrow (BM) niche. Both human BCL2 and MCL1 protein expression co-localized with engrafted human leukemic CD34+ cells in the bone marrow epiphysis and served as important biomarkers of response to sabutoclax. Importantly, intravenous treatment with sabutoclax reduced BC CML CSC survival in both marrow and splenic niches at doses that spared normal hematopoietic stem cells in RAG2-/-gamma c-/- xenograft models established with cord blood CD34+ cells. While dasatinib treatment alone increased serially transplantable quiescent BC CML CSC in BM, sabutoclax decreased CSC survival commensurate with upregulation of short pro-apoptotic and downregulation of long anti-apopoptotic BCL2 family isoforms. While previous studies involved intraperitoneal administration, in the last 12 months we have focused on a more clinically relevant intravenous (IV) administration schedule with IV sabutoclax administered alone or in combination with oral dasatinib. In these studies, sabutoclax sensitized quiescent CSC to dasatinib resulting in a significant decrease in CSC survival versus dasatinib alone. Moreover, mice serially transplanted with human cells from combination treated mice had increased survival compared to serial transplants of single agent treated tissues. Human CD34+ cells from the BM of combination treated mice had more cells in cycle than CD34+ cells compared to the BM of mice treated with dasatinib alone. The frequency of CD34+BCL2+ and CD34+MCL1+ BC CSC were significantly lower in BM treated with a combination of sabutoclax and dasatinib suggesting that the combination acts synergistically to decrease CSC survival and increase the lifespan of CSC engrafted mice. During this 12-month reporting period, sabutoclax production was successfully scaled up by two separate CMOs, Syncom and Norac. Dr. Pellecchia (SBMRI) provided flash chromatography purified sabutoclax to Dr. Jamieson for use in cellular and in vivo studies in addition to conducting QC analyses (integrity and purity) on scaled up sabutoclax formulations produced by Norac (4g) and Syncom (30g) in different vehicles. In formulation studies, a flash chromatography method was developed and qualified that separates impurities and degradation compounds from sabutoclax. Additional solubility and stability studies were performed to identify an IV Solutol formulation, compared with the previous IP DMSO/PBS Tween formulation, which could be used for both pre-clinical studies and in future clinical trials. Pilot PK studies in mice and rats were conducted with the Solutol formulated sabutoclax and showed weight loss associated with impurities that could be readily removed by standard flash chromatography. As a result, ssabutoclax production will include flash chromatography to enhance purity and stability and this material will be used for further PK and PD studies. In conclusion, we are on track to accomplish our milestones as set forth in the grant and anticipate that sabutoclax will form the basis of combination clinical studies aimed at eradicating quiescent CSC in a broad array of refractory malignancies.

Recent cancer stem cell research performed by ourselves and others has bolstered interest in BCL2 family member expression and inhibition in chronic myeloid leukemia (CML), acute myeloid leukemia (AML) and breast cancer (Goff DJ et al Cell Stem Cell 2013; Lagadinou ED et al Cell Stem Cell 2013; Vaillant F et al Cancer Cell 2013). Overexpression of pro-survival BCL2 family genes has been linked to therapeutic resistance driven by dormant, self-renewing CSC. Thus, the BCL2 family represents an attractive therapeutic target that may provide the potential to reduce relapse rates. Because of the greater proclivity for alternative splicing in humans compared with mice, our CIRM ETll funded research has focused on whole transcriptome RNA sequencing, splice isoform specific qRT-PCR and BCL2 PCR array analysis of FACS-purified CSC from patients with CML and CSC derived from human blast crisis CML engrafted RAG2-/-gc-/- mouse models. A Pan-BCL2 inhibitor renders bone-marrow-resident human leukemia stem cells sensitive to tyrosine kinase inhibition. Cell Stem Cell. 2013 Mar 7;12(3):316-28) was featured in a lead article in Cell Stem Cell in March. This study also led to a number of disclosures relating to unique self-renewal and survival gene splice isoform based CSC detection and patient prognostication strategies. As a result, pan BCL2 targeting has generated considerable interest from academic and pharmaceutical investigators who would like to adopt the approach of dormant CSC sensitization to agents that target dividing cells, including tyrosine kinase inhibitors, chemotherapy and radiation therapy.