Summary Leukemia stem cells (LSC) play a pivotal role in chronic myeloid leukemia (CML) tyrosine kinase inhibitor (TKI) resistance and progression to blast crisis (BC), in part, through alternative splicing of self-renewal and survival genes. To elucidate splice isoform regulators of human BC LSC maintenance, we performed whole transcriptome RNA sequencing; splice isoform-specific qRT-PCR, nanoproteomics, stromal co-culture and BC LSC xenotransplantation analyses. Cumulatively, these studies show that alternative splicing of multiple pro-survival BCL2 family genes promotes malignant transformation of myeloid progenitors into BC LSC that are quiescent in the marrow niche and contribute to therapeutic resistance. Notably, a novel pan-BCL2 inhibitor, sabutoclax, renders marrow niche-resident BC LSC sensitive to TKIs at doses that spare normal progenitors. These findings underscore the importance of alternative BCL2 family splice isoform expression in BC LSC maintenance and suggest that combinatorial inhibition of pro-survival BCL2 family proteins and BCR-ABL may eliminate dormant LSC and obviate resistance.

The transcription factor Twist1 induces Epithelial-Mesenchymal Transition and extracellular matrix degradation to promote tumor metastasis. Although Twist1 also plays a role in embryonic vascular development and tumor angiogenesis, the molecular mechanisms that underlie these processes are not as well understood. Here, we report a novel function for Twist1 in modifying the tumor microenvironment to promote progression. We found that expression of Twist1 in human mammary epithelial cells potently promoted angiogenesis. Surprisingly, Twist1 expression did not increase the secretion of the common pro-angiogenic factors VEGF and bFGF, but rather induced expression of the macrophage chemoattractant CCL2. Attenuation of endogenous Twist1 in vivo blocked macrophage recruitment and angiogenesis, whereas exogenous CCL2 rescued the ability of tumor cells lacking Twist1 to attract macrophages and promote angiogenesis. Macrophage recruitment also was essential for the ability of Twist1-expressing cells to elicit a strong angiogenic response. Together, our findings show how Twist1 recruits stromal macrophages through CCL2 induction to promote angiogenesis and tumor progression. Since Twist1 expression has been associated with poor survival in many human cancers, this finding suggests that anti-CCL2 therapy may offer a rational strategy to treat Twist1-positive metastatic cancers.

Aquatic ecosystems around the world face serious threats from anthropogenic contaminants. Results from 8 years of field and laboratory investigations indicate that sublethal contaminant exposure is occurring in the early life stages of striped bass in the San Francisco Estuary, a population in continual decline since its initial collapse during the 1970s. Biologically significant levels of polychlorinated biphenyls, polybrominated diphenyl ethers, and current-use/legacy pesticides were found in all egg samples from river-collected fish. Developmental changes previously unseen with standard methods were detected with a technique using the principles of unbiased stereology. Abnormal yolk utilization, brain and liver development, and overall growth were observed in larvae from river-collected fish. Histopathological analyses confirmed and identified developmental alterations. Using this methodology enabled us to present a conclusive line of evidence for the maternal transfer of xenobiotics and their adverse effects on larval striped bass in this estuary.Morone saxatilis ͉ contaminants ͉ biomarkers ͉ histopathology ͉ unbiased stereology

3739 Leukemia stem cells (LSC) represent a frequently dormant self-renewing population integral to the initiation, maintenance, and progression of human chromic myeloid leukemia (CML). The current standard of care dasatinib, a BCR-ABL targeted tyrosine kinase inhibitor (TKI), effectively eradicates the bulk of CML cells but frequently fails to affect the LSC population that is thought to drive CML relapse. Members in the BCL2 family are proteins that regulate apoptosis, 6 of which regulate cell survival. Each of these 6 members has a long and short isoform with opposing functions; generally, long isoforms promote cell survival while the short isoforms promote apoptosis. Previously, we demonstrated that upregulation of pro-survival BCL2 proteins in CML LSC contributes to chemotherapy resistance and LSC quiescence in protective hematopoietic niches. LSC found in different hematopoietic niches differ in their response to TKI treatment. Niche affects LSC cell cycle, either by maintaining quiescence or by promoting rapid cell cycling. Quiescent cells are a hurdle for traditional chemotherapy, which usually targets rapidly cycling cells, leaving the quiescent LSC untouched. We hypothesize that the inhibition of pro-survival BCL2 protein family members will sensitize LSC to dasatinib therapy and therefore prevent CML relapse. We tested a novel pan pro-survival BCL2 family protein inhibitor, sabutoclax, delivered by intravenous injection either alone or in combination with oral dasatinib in immunodeficient RAG2−/-gc−/- mice engrafted with BC CML patient samples. After treatment, LSC burden, self-renewal, and cell cycle status were quantified using FACS. Our results showed a reduction in the LSC burden in combination treated mice when compared to mice that received either drug alone. Mice treated with the combination regimen were found to have fewer quiescent human leukemic cells than their counterparts that received single agent treatments. Immunofluorescence staining confirmed the reduction of quiescent cells in the bone marrow after combination treatment when compared to single agent or vehicle treatments. We validated the molecular targets by using human specific splice isoform primers to perform RT-qPCR on FACS sorted LSC and showed a reduction in the BCL2 long to short isoform ratio in sabutoclax versus vehicle treated animals, indicating a skewing towards the pro-apoptotic splice variant. Together, these results indicate that the combination strategy with a pan pro-survival BCL2 family inhibitor and a tyrosine kinase inhibitor may be the foundation for a promising clinical strategy to effectively eliminate LSC and prevent cancer progression and relapse. Disclosures: No relevant conflicts of interest to declare.