Repression of Mcl-1 expression by the CDC7/CDK9 inhibitor PHA-767491 overcomes bone marrow stroma-mediated drug resistance in AML.

Acute myeloid leukaemia (AML) is an aggressive cancer with 50-75% of patients relapsing even after successful chemotherapy. The role of the bone marrow microenvironment (BMM) in protecting AML cells from chemotherapeutics and causing consequent relapse is increasingly recognised. However the role that the anti-apoptotic Bcl-2 proteins play as effectors of BMM-mediated drug resistance are less understood. Here we show that bone marrow mesenchymal stromal cells (BMSC) provide resistance to AML cells against BH3-mimetics, cytarabine and daunorubicin, but this is not mediated by Bcl-2 and/or Bcl-XL as previously thought. Instead, BMSCs induced Mcl-1 expression over Bcl-2 and/or Bcl-XL in AML cells and inhibition of Mcl-1 with a small-molecule inhibitor, A1210477, or repressing its expression with the CDC7/CDK9 dual-inhibitor, PHA-767491 restored sensitivity to BH3-mimetics. Furthermore, combined inhibition of Bcl-2/Bcl-XL and Mcl-1 could revert BMSC-mediated resistance against cytarabine + daunorubicin. Importantly, the CD34+/CD38- leukemic stem cell-encompassing population was equally sensitive to the combination of PHA-767491 and ABT-737. These results indicate that Bcl-2/Bcl-XL and Mcl-1 act in a redundant fashion as effectors of BMM-mediated AML drug resistance and highlight the potential of Mcl-1-repression to revert BMM-mediated drug resistance in the leukemic stem cell population, thus, prevent disease relapse and ultimately improve patient survival.

The BH3-mimetic ABT-737 effectively kills acute myeloid leukemia initiating cells.

The anti-apoptotic proteins Bcl-XL and Bcl-2 are abundantly expressed in hematopoietic stem cells and/or progenitor cells. Furthermore, leukemic cells expressing these proteins are enriched in minimal residual disease cell populations. This prompted us to test the BH3-mimetic compound ABT-737 for its ability to eradicate putative leukemic stem cells. ABT-737 demonstrated potent cytotoxic effects in all patient samples tested. The efficacy of ABT-737 against AML blasts and the primitive CD34(+)/CD38(-) population was equal and independent of sensitivity to cytarabine/daunorubicin. These results, together with previously reported synergistic effects of ABT-737 with chemotherapeutics make BH3-mimetics promising candidates for future AML treatment regimens.

Impaired SLAM-SLAM homotypic interaction between invariant NKT cells and dendritic cells affects differentiation of IL-4/IL-10-secreting NKT2 cells in nonobese diabetic mice.

The regulatory function of invariant NKT (iNKT) cells for tolerance induction and prevention of autoimmunity is linked to a specific cytokine profile that comprises the secretion of type 2 cytokines like IL-4 and IL-10 (NKT2 cytokine profile). The mechanism responsible for iNKT cell differentiation toward a type 2 phenotype is unknown. Herein we show that costimulatory signals provided by the surface receptor signaling lymphocytic activation molecule (SLAM) on myeloid dendritic cells (mDC) to iNKT cells is crucial for NKT2 orientation. Additionally, we demonstrate that the impaired acquisition of an NKT2 cytokine phenotype in nonobese diabetic (NOD) mice that spontaneously develop autoimmune diabetes is due to defective SLAM-induced signals generated by NOD mDC. Mature mDC of C57BL/6 mice express SLAM and induce C57BL/6 or NOD iNKT cells to acquire a predominant NKT2 cytokine phenotype in response to antigenic stimulation with the iNKT cell-specific Ag, the alpha-galactosylceramide. In contrast, mature NOD mDC express significantly lower levels of SLAM and are unable to promote GATA-3 (the SLAM-induced intracellular signal) up-regulation and IL-4/IL-10 production in iNKT cells from NOD or C57BL/6 mice. NOD mice carry a genetic defect of the Slamf1 gene that is associated with reduced SLAM expression on double-positive thymocytes and altered iNKT cell development in the thymus. Our data suggest that the genetic Slamf1 defect in NOD mice also affects SLAM expression on other immune cells such as the mDC, thus critically impairing the peripheral differentiation of iNKT cells toward a regulatory NKT2 type.

Distinct homeostatic requirements of CD4+ and CD4- subsets of Valpha24-invariant natural killer T cells in humans.

CD1d-restricted Valpha24-invariant natural killer T cells (iNKTs) are important in immunoregulation. CD4(+) and CD4(-) iNKTs develop with similar frequencies in murine thymus and depend on interleukin-15 (IL-15) in periphery. However, homeostatic requirements of iNKTs have not been analyzed in humans. We evaluated thymic production, peripheral dynamics, and functional maturation of human iNKTs. CD4(+) subset comprises 90% of iNKTs in mature thymocytes and cord blood (CB) but only 40% in adult blood. Using T-cell receptor excision circle (TREC) analysis, we directly measured in vivo replicative history of CD4(+) and CD4(-) iNKT cells. Compared to CD4(+), CD4(-) iNKTs contain fewer TRECs, express higher levels of IL-2Rbeta, and proliferate with higher rate in response to IL-15. In contrast, CD4(+) cells express higher levels of IL-7Ralpha and better respond to IL-7. Neither thymic nor CB iNKTs are able to produce cytokines unless they are induced to proliferate. Therefore, unlike in the mouse, human CD4(+) iNKTs are mainly supported by thymic output and limited peripheral expansion, whereas CD4(-) cells undergo extensive peripheral expansion, and both subsets develop their functions in periphery. These findings reveal important differences in homeostatic requirements and functional maturation between murine and human iNKTs that are to be considered for clinical purposes.