GLI2 inhibition abrogates human leukemia stem cell dormancy.

BACKGROUND: Dormant leukemia stem cells (LSC) promote therapeutic resistance and leukemic progression as a result of unbridled activation of stem cell gene expression programs. Thus, we hypothesized that 1) deregulation of the hedgehog (Hh) stem cell self-renewal and cell cycle regulatory pathway would promote dormant human LSC generation and 2) that PF-04449913, a clinical antagonist of the GLI2 transcriptional activator, smoothened (SMO), would enhance dormant human LSC eradication. METHODS: To test these postulates, whole transcriptome RNA sequencing (RNA-seq), microarray, qRT-PCR, stromal co-culture, confocal fluorescence microscopic, nanoproteomic, serial transplantation and cell cycle analyses were performed on FACS purified normal, chronic phase (CP) chronic myeloid leukemia (CML), blast crisis (BC) phase CML progenitors with or without PF-04449913 treatment. RESULTS: Notably, RNA-seq analyses revealed that Hh pathway and cell cycle regulatory gene overexpression correlated with leukemic progression. While lentivirally enforced GLI2 expression enhanced leukemic progenitor dormancy in stromal co-cultures, this was not observed with a mutant GLI2 lacking a transactivation domain, suggesting that GLI2 expression prevented cell cycle transit. Selective SMO inhibition with PF-04449913 in humanized stromal co-cultures and LSC xenografts reduced downstream GLI2 protein and cell cycle regulatory gene expression. Moreover, SMO inhibition enhanced cell cycle transit and sensitized BC LSC to tyrosine kinase inhibition in vivo at doses that spare normal HSC. CONCLUSION: In summary, while GLI2, forms part of a core HH pathway transcriptional regulatory network that promotes human myeloid leukemic progression and dormant LSC generation, selective inhibition with PF-04449913 reduces the dormant LSC burden thereby providing a strong rationale for clinical trials predicated on SMO inhibition in combination with TKIs or chemotherapeutic agents with the ultimate aim of obviating leukemic therapeutic resistance, persistence and progression.

Impacts of Marine Corps Body Composition and Military Appearance Program (BCMAP) Standards on Individual Outcomes and Talent Management.

The U.S. Marine Corps' Body Composition and Military Appearance Program (BCMAP) standards were not developed from populations that reflect the current makeup of the force and the fitness requirements that they are subject to. Research suggests that the implementation of these standards could drive marines to adopt unhealthy behaviors, primarily those associated with disordered eating, to meet the standards while disproportionately affecting communities of color and women more generally. Furthermore, these unhealthy behaviors can cause significant short- and long-term mental and physical health problems that could negatively affect individual marines during their service and long after. Although there is some limited research on body-composition standards and eating disorders in the services, there has been little assessment of how the negative effects of policy and the behaviors associated with it affect the mental and physical health of individual marines (particularly those from communities of color and women more generally), career retention, and overall military readiness. Service and U.S. Department of Defense leadership have made talent management and diversity of the force at different levels of leadership an institutional priority; understanding how the BCMAP affects the force will help meet these objectives. This research will help decisionmakers understand how the BCMAP and its associated policies drive individual behavior, particularly for women in general and communities of color. It will also inform talent-management efforts and discussions about relevant national security implications while providing recommendations and a general framework for policy change.

GS2 as a retinol transacylase and as a catalytic dyad independent regulator of retinylester accretion.

GS2 (PNPLA4; iPLAeta) is the smallest member of the patatin-like family of phospholipases (PNPLA). It was initially identified by its ability to hydrolyze retinylesters (RE) in cell homogenates, and was later found to esterify retinol using a variety of acyl donors. In the present study we set out to determine its cellular function and examined its impact on RE status in 293T cells transfected with GS2, GS2-M1 (a non-translatable mutant of GS2) and empty vector, in fibroblasts isolated from normal and GS2-null donors and in SCC12b and in a somatic cell knock-out of GS2 (SCC12b-GS2(neo/-)), that we generated by homologous recombination. At 50nM medium retinol, GS2 had no significant impact on RE accumulation. However, at 2muM retinol, GS2 promoted a 1.6- to 5-fold increase in RE accumulation. To verify role of GS2 as a catalyst, RE levels were measured in 293T transfected wild type GS2, catalytic dyad mutants devoid of enzymatic activity, or alanine substitution mutants spanning the entire GS2 sequence. Surprisingly, every GS2 mutant promoted RE accumulation. This activity was also observed in the GS2 paralogues and rat orthologue. The data demonstrate that within the context of the cell GS2 promotes RE accumulation and may do so either as a catalyst or as a regulatory protein that enhances RE formation catalyzed by other acyl transferases.

A Pan-BCL2 inhibitor renders bone-marrow-resident human leukemia stem cells sensitive to tyrosine kinase inhibition.

Leukemia stem cells (LSCs) play a pivotal role in the resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors (TKIs) and its progression to blast crisis (BC), in part, through the 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 quantitative RT-PCR (qRT-PCR), nanoproteomics, stromal coculture, and BC LSC xenotransplantation analyses. Cumulatively, these studies show that the alternative splicing of multiple prosurvival BCL2 family genes promotes malignant transformation of myeloid progenitors into BC LSCS that are quiescent in the marrow niche and that contribute to therapeutic resistance. Notably, sabutoclax, a pan-BCL2 inhibitor, renders marrow-niche-resident BC LSCs 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 the combinatorial inhibition of prosurvival BCL2 family proteins and BCR-ABL may eliminate dormant LSCs and obviate resistance.

Optical imaging of progenitor cell homing to patient-derived tumors.

Capitalizing on cellular homing to cancer is a promising strategy for targeting malignant cells for diagnostic, monitoring and therapeutic purposes. Murine C17.2 neural progenitor cells (NPC) demonstrate a tropism for cell line-derived tumors, but their affinity for patient-derived tumors is unknown. We tested the hypothesis that NPC accumulate in patient-derived tumors at levels detectable by optical imaging. Mice bearing solid tumors after transplantation with patient-derived leukemia cells and untransplanted controls received 10(6) fluorescent DiR-labeled NPC daily for 1-4 days, were imaged, then sacrificed. Tissues were analyzed by immunofluorescence and flow cytometry to detect tumor cell engraftment (CD45) and NPC (FITC-ß galactosidase or DiR). Tumors consisted primarily of CD45-positive cells and demonstrated mild fluorescence, corresponding to frequent clusters of FITC-ß gal-positive cells. Both transplanted and control mice demonstrated the highest fluorescent signal in the spleens and other tissues of the reticuloendothelial activating system. However, only rare FITC-ß gal-positive cells were detected in the mildly engrafted transplanted spleens and none in the control spleens, suggesting that their high DiR signal reflects the sequestration of DiR-positive debris. The mildly engrafted transplanted kidneys demonstrated low fluorescent signal and rare FITC-ß gal-positive cells whereas control kidneys were negative. Results indicate that NPC accumulate in tissues containing patient-derived tumor cells in a manner that is detectable by ex vivo optical imaging and proportional to the level of tumor engraftment, suggesting a capacity to home to micrometastatic disease. As such, NPC could have significant clinical applications for the targeted diagnosis and treatment of cancer.

NOTCH1 signaling promotes human T-cell acute lymphoblastic leukemia initiating cell regeneration in supportive niches.

BACKGROUND: Leukemia initiating cells (LIC) contribute to therapeutic resistance through acquisition of mutations in signaling pathways, such as NOTCH1, that promote self-renewal and survival within supportive niches. Activating mutations in NOTCH1 occur commonly in T cell acute lymphoblastic leukemia (T-ALL) and have been implicated in therapeutic resistance. However, the cell type and context specific consequences of NOTCH1 activation, its role in human LIC regeneration, and sensitivity to NOTCH1 inhibition in hematopoietic microenvironments had not been elucidated. METHODOLOGY AND PRINCIPAL FINDINGS: We established humanized bioluminescent T-ALL LIC mouse models transplanted with pediatric T-ALL samples that were sequenced for NOTCH1 and other common T-ALL mutations. In this study, CD34(+) cells from NOTCH1(Mutated) T-ALL samples had higher leukemic engraftment and serial transplantation capacity than NOTCH1(Wild-type) CD34(+) cells in hematopoietic niches, suggesting that self-renewing LIC were enriched within the NOTCH1(Mutated) CD34(+) fraction. Humanized NOTCH1 monoclonal antibody treatment reduced LIC survival and self-renewal in NOTCH1(Mutated) T-ALL LIC-engrafted mice and resulted in depletion of CD34(+)CD2(+)CD7(+) cells that harbor serial transplantation capacity. CONCLUSIONS: These results reveal a functional hierarchy within the LIC population based on NOTCH1 activation, which renders LIC susceptible to targeted NOTCH1 inhibition and highlights the utility of NOTCH1 antibody targeting as a key component of malignant stem cell eradication strategies.