Daratumumab (Dara), a multiple myeloma (MM) therapy, is an antibody against the surface receptor CD38, which is expressed not only on plasma cells but also on NK cells and monocytes. Correlative data ...have highlighted the immune-modulatory role of Dara, despite the paradoxical observation that Dara regimens decrease the frequency of total NK cells. Here we show that, despite this reduction, NK cells play a pivotal role in Dara anti-MM activity. CD38 on NK cells is essential for Dara-induced immune modulation, and its expression is restricted to NK cells with effector function. We also show that Dara induces rapid CD38 protein degradation associated with NK cell activation, leaving an activated CD38-negative NK cell population. CD38+ NK cell targeting by Dara also promotes monocyte activation, inducing an increase in T-cell costimulatory molecules (CD86/80) and enhancing anti-MM phagocytosis activity ex vivo and in vivo. In support of Dara's immunomodulating role, we show that MM patients that discontinued Dara therapy because of progression maintain targetable unmutated surface CD38 expression on their MM cells, but retain effector cells with impaired cellular immune function. In summary, we report that CD38+ NK cells may be an unexplored therapeutic target for priming the immune system of MM patients.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Leukemia stem cells (LSCs) in individuals with chronic myelogenous leukemia (CML) (hereafter referred to as CML LSCs) are responsible for initiating and maintaining clonal hematopoiesis. These cells ...persist in the bone marrow (BM) despite effective inhibition of BCR-ABL kinase activity by tyrosine kinase inhibitors (TKIs). Here we show that although the microRNA (miRNA) miR-126 supported the quiescence, self-renewal and engraftment capacity of CML LSCs, miR-126 levels were lower in CML LSCs than in long-term hematopoietic stem cells (LT-HSCs) from healthy individuals. Downregulation of miR-126 levels in CML LSCs was due to phosphorylation of Sprouty-related EVH1-domain-containing 1 (SPRED1) by BCR-ABL, which led to inhibition of the RAN-exportin-5-RCC1 complex that mediates miRNA maturation. Endothelial cells (ECs) in the BM supply miR-126 to CML LSCs to support quiescence and leukemia growth, as shown using mouse models of CML in which Mir126a (encoding miR-126) was conditionally knocked out in ECs and/or LSCs. Inhibition of BCR-ABL by TKI treatment caused an undesired increase in endogenous miR-126 levels, which enhanced LSC quiescence and persistence. Mir126a knockout in LSCs and/or ECs, or treatment with a miR-126 inhibitor that targets miR-126 expression in both LSCs and ECs, enhanced the in vivo anti-leukemic effects of TKI treatment and strongly diminished LSC leukemia-initiating capacity, providing a new strategy for the elimination of LSCs in individuals with CML.
The FLT3-ITD mutation is frequently observed in acute myeloid leukemia (AML) and is associated with poor prognosis. In such patients, FLT3 tyrosine kinase inhibitors (TKIs) are only partially ...effective and do not eliminate the leukemia stem cells (LSCs) that are assumed to be the source of treatment failure. Here, we show that the NAD-dependent SIRT1 deacetylase is selectively overexpressed in primary human FLT3-ITD AML LSCs. This SIRT1 overexpression is related to enhanced expression of the USP22 deubiquitinase induced by c-MYC, leading to reduced SIRT1 ubiquitination and enhanced stability. Inhibition of SIRT1 expression or activity reduced the growth of FLT3-ITD AML LSCs and significantly enhanced TKI-mediated killing of the cells. Therefore, these results identify a c-MYC-related network that enhances SIRT1 protein expression in human FLT3-ITD AML LSCs and contributes to their maintenance. Inhibition of this oncogenic network could be an attractive approach for targeting FLT3-ITD AML LSCs to improve treatment outcomes.
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•SIRT1 protein expression is selectively enhanced in FLT3-ITD AML stem cells•Combined SIRT1 and FLT3 inhibition increases elimination of FLT3-ITD AML stem cells•SIRT1 overexpression in FLT3-ITD AML is related to c-MYC activation•c-MYC increases SIRT1 expression by enhancing expression of the USP22 deubiquitinase
Li et al. find that c-MYC via USP22 deubiquitinase enhances SIRT1 expression and promotes maintenance of human AML stem cells, which suggests a strategy to target leukemia stem cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Myelodysplastic syndrome (MDS), a largely incurable hematological malignancy, is derived from aberrant clonal hematopoietic stem/progenitor cells (HSPCs) that persist after conventional therapies. ...Defining the mechanisms underlying MDS HSPC maintenance is critical for developing MDS therapy. The deacetylase SIRT1 regulates stem cell proliferation, survival, and self-renewal by deacetylating downstream proteins. Here we show that SIRT1 protein levels were downregulated in MDS HSPCs. Genetic or pharmacological activation of SIRT1 inhibited MDS HSPC functions, whereas SIRT1 deficiency enhanced MDS HSPC self-renewal. Mechanistically, the inhibitory effects of SIRT1 were dependent on TET2, a safeguard against HSPC transformation. SIRT1 deacetylated TET2 at conserved lysine residues in its catalytic domain, enhancing TET2 activity. Our genome-wide analysis identified cancer-related genes regulated by the SIRT1/TET2 axis. SIRT1 activation also inhibited functions of MDS HSPCs from patients with TET2 heterozygous mutations. Altogether, our results indicate that restoring TET2 function through SIRT1 activation represents a promising means to target MDS HSPCs.
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•SIRT1 depletion enhances MDS HSPC self-renewal and growth•SIRT1 insufficiency in MDS promotes TET2 hyperacetylation•SIRT1 agonist treatment blocks MDS HSPC maintenance•SIRT1 activation deacetylates TET2 and enhances its catalytic activity
An improved understanding of the mechanisms regulating myelodysplastic syndrome (MDS) hematopoietic stem/progenitor cell (HSPC) growth and self-renewal is critical for developing MDS therapy. Sun et al. report that SIRT1 deficiency-induced TET2 hyperacetylation promotes MDS HSPC function and provides an approach to target MDS HSPCs by activating SIRT1 deacetylase.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Acute myeloid leukemia (AML) is driven and sustained by leukemia stem cells (LSCs) with unlimited self-renewal capacity and resistance to chemotherapy. Mutation in the TP53 tumor suppressor is ...relatively rare in de novo AML; however, p53 can be regulated through post-translational mechanisms. Here, we show that p53 activity is inhibited in inv(16)+ AML LSCs via interactions with the CBFβ-SMMHC (CM) fusion protein and histone deacetylase 8 (HDAC8). HDAC8 aberrantly deacetylates p53 and promotes LSC transformation and maintenance. HDAC8 deficiency or inhibition using HDAC8-selective inhibitors (HDAC8i) effectively restores p53 acetylation and activity. Importantly, HDAC8 inhibition induces apoptosis in inv(16)+ AML CD34+ cells, while sparing the normal hematopoietic stem cells. Furthermore, in vivo HDAC8i administration profoundly diminishes AML propagation and abrogates leukemia-initiating capacity of both murine and patient-derived LSCs. This study elucidates an HDAC8-mediated p53-inactivating mechanism promoting LSC activity and highlights HDAC8 inhibition as a promising approach to selectively target inv(16)+ LSCs.
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•CBFβ-SMMHC (CM) forms an aberrant protein complex with p53 and HDAC8•HDAC8 promotes CM-mediated LSC transformation by aberrantly deacetylating p53•HDAC8 inhibition selectively targets inv(16)+ AML CD34+ cells by reactivating p53•Inhibition of HDAC8 eliminates AML propagation and LSC leukemia-initiating activity
Qi et al. demonstrate that the inv(16) fusion protein interacts with the tumor suppressor p53 and HDAC8, thereby causing aberrant HDAC8-mediated deacetylation and inactivation of p53. HDAC8 deletion or inhibition selectively depletes LSC activity by reactivating p53, highlighting HDAC8 as an effective therapeutic target to specifically eliminate inv(16) AML LSCs.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The SRC family kinases (SFKs) and the receptor tyrosine kinase c-Kit are activated in human acute myeloid leukemia (AML) cells. We show here that the SFKs LYN, HCK, or FGR are overexpressed and ...activated in AML progenitor cells. Treatment with the SFK and c-KIT inhibitor dasatinib selectively inhibits human AML stem/progenitor cell growth in vitro. Importantly, dasatinib markedly increases the elimination of AML stem cells capable of engrafting immunodeficient mice by chemotherapeutic agents. In vivo dasatinib treatment enhances chemotherapy-induced targeting of primary murine AML stem cells capable of regenerating leukemia in secondary recipients. Our studies suggest that enhanced targeting of AML cells by the combination of dasatinib with daunorubicin may be related to inhibition of AKT-mediated human mouse double minute 2 homolog phosphorylation, resulting in enhanced p53 activity in AML cells. Combined treatment using dasatinib and chemotherapy provides a novel approach to increasing p53 activity and enhancing targeting of AML stem cells.
•SRC family kinases are activated in AML stem/progenitor cells and contribute to AML stem cell survival and proliferation.•Combined inhibition of SFKs and c-KIT with dasatinib enhances p53-mediated elimination of AML stem cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
SHP2, a cytoplasmic protein-tyrosine phosphatase encoded by the PTPN11 gene, plays a critical role in developmental hematopoiesis in the mouse, and gain-of-function mutations of SHP2 are associated ...with hematopoietic malignancies. However, the role of SHP2 in adult hematopoiesis has not been addressed in previous studies. In addition, the role of SHP2 in human hematopoiesis has not been described. These questions are of considerable importance given the interest in development of SHP2 inhibitors for cancer treatment. We used shRNA-mediated inhibition of SHP2 expression to investigate the function of SHP2 in growth factor (GF) signaling in normal human CD34+ cells. SHP2 knockdown resulted in markedly reduced proliferation and survival of cells cultured with GF, and reduced colony-forming cell growth. Cells expressing gain-of-function SHP2 mutations demonstrated increased dependency on SHP2 expression for survival compared with cells expressing wild-type SHP2. SHP2 knockdown was associated with significantly reduced myeloid and erythroid differentiation with retention of CD34+ progenitors with enhanced proliferative capacity. Inhibition of SHP2 expression initially enhanced and later inhibited STAT5 phosphorylation and reduced expression of the antiapoptotic genes MCL1 and BCLXL. These results indicate an important role for SHP2 in STAT5 activation and GF-mediated proliferation, survival, and differentiation of human progenitor cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Daratumumab (Dara), a human immunoglobulin G1 kappa (IgG1κ) monoclonal anti-CD38 antibody, has been approved by the U.S. Food and Drug Administration for the treatment of relapsed multiple myeloma ...(MM) as a single agent as well as in combination with immunomodulatory drugs (IMiDs) and proteasome inhibitors (PI). Although the scientific rationale behind the use of Dara in combination with IMiDs has been extensively explored, the molecular mechanisms underlying Dara-PI regimens have not yet been investigated. Here, we demonstrate that CD38 on the surface of MM cells is rapidly internalized after Dara treatment; we also show that Dara treatment impairs MM cell adhesion, an effect that can be rescued by using the endocytosis inhibitor Dynasore. Finally, we show that Dara potentiates bortezomib (BTZ) killing of MM cells in vitro and in vivo, independent of its function as an immune activator. In conclusion, our data show that Dara impairs MM cell adhesion, which results in an increased sensitivity of MM to proteasome inhibition.
BackgroundType I interferons (IFN-Is), secreted by hematopoietic cells, drive immune surveillance of solid tumors. However, the mechanisms of suppression of IFN-I-driven immune responses in ...hematopoietic malignancies including B-cell acute lymphoblastic leukemia (B-ALL) are unknown.MethodsUsing high-dimensional cytometry, we delineate the defects in IFN-I production and IFN-I-driven immune responses in high-grade primary human and mouse B-ALLs. We develop natural killer (NK) cells as therapies to counter the intrinsic suppression of IFN-I production in B-ALL.ResultsWe find that high expression of IFN-I signaling genes predicts favorable clinical outcome in patients with B-ALL, underscoring the importance of the IFN-I pathway in this malignancy. We show that human and mouse B-ALL microenvironments harbor an intrinsic defect in paracrine (plasmacytoid dendritic cell) and/or autocrine (B-cell) IFN-I production and IFN-I-driven immune responses. Reduced IFN-I production is sufficient for suppressing the immune system and promoting leukemia development in mice prone to MYC-driven B-ALL. Among anti-leukemia immune subsets, suppression of IFN-I production most markedly lowers the transcription of IL-15 and reduces NK-cell number and effector maturation in B-ALL microenvironments. Adoptive transfer of healthy NK cells significantly prolongs survival of overt ALL-bearing transgenic mice. Administration of IFN-Is to B-ALL-prone mice reduces leukemia progression and increases the frequencies of total NK and NK-cell effectors in circulation. Ex vivo treatment of malignant and non-malignant immune cells in primary mouse B-ALL microenvironments with IFN-Is fully restores proximal IFN-I signaling and partially restores IL-15 production. In B-ALL patients, the suppression of IL-15 is the most severe in difficult-to-treat subtypes with MYC overexpression. MYC overexpression promotes sensitivity of B-ALL to NK cell-mediated killing. To counter the suppressed IFN-I-induced IL-15 production in MYChigh human B-ALL, we CRISPRa-engineered a novel human NK-cell line that secretes IL-15. CRISPRa IL-15-secreting human NK cells kill high-grade human B-ALL in vitro and block leukemia progression in vivo more effectively than NK cells that do not produce IL-15.ConclusionWe find that restoration of the intrinsically suppressed IFN-I production in B-ALL underlies the therapeutic efficacy of IL-15-producing NK cells and that such NK cells represent an attractive therapeutic solution for the problem of drugging MYC in high-grade B-ALL.
Background
Myelodysplastic syndrome (MDS) arises from a rare population of aberrant hematopoietic stem and progenitor cells (HSPCs). These cells are relatively quiescent and therefore treatment ...resistant. Understanding mechanisms underlying their maintenance is critical for effective MDS treatment.
Methods
We evaluated microRNA‐126 (miR‐126) levels in MDS patients’ sample and in a NUP98‐HOXD13 (NHD13) murine MDS model along with their normal controls and defined its role in MDS HSPCs’ maintenance by inhibiting miR‐126 expression in vitro and in vivo. Identification of miR‐126 effectors was conducted using biotinylated miR‐126 pulldown coupled with transcriptome analysis. We also tested the therapeutic activity of our anti‐miR‐126 oligodeoxynucleotide (miRisten) in human MDS xenografts and murine MDS models.
Results
miR‐126 levels were higher in bone marrow mononuclear cells from MDS patients and NHD13 mice relative to their respective normal controls (P < 0.001). Genetic deletion of miR‐126 in NHD13 mice decreased quiescence and self‐renewal capacity of MDS HSPCs, and alleviated MDS symptoms of NHD13 mice. Ex vivo exposure to miRisten increased cell cycling, reduced colony‐forming capacity, and enhanced apoptosis in human MDS HSPCs, but spared normal human HSPCs. In vivo miRisten administration partially reversed pancytopenia in NHD13 mice and blocked the leukemic transformation (combination group vs DAC group, P < 0.0001). Mechanistically, we identified the non‐coding RNA PTTG3P as a novel miR‐126 target. Lower PTTG3P levels were associated with a shorter overall survival in MDS patients.
Conclusions
MiR‐126 plays crucial roles in MDS HSPC maintenance. Therapeutic targeting of miR‐126 is a potentially novel approach in MDS.
MiR‐126 levels are higher in stem/progenitor cells from MDS patients relative to normal counterparts.
MiR‐126 KO inhibits MDS stem/progenitor cell maintenance.
In vivo administration of a miR‐126 inhibitor in combination with decitabine partially reverses pancytopenia in a murine MDS model and decreases the risk of leukemic transformation.
The non‐coding RNA PTTG3P is a downstream target of miR‐126 in MDS cells.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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