The development and function of stem and progenitor cells that produce blood cells are vital in physiology. GATA2 mutations cause GATA-2-deficiency syndrome involving immunodeficiency, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). GATA-2 physiological activities necessitate that it be strictly regulated, and cell type-specific enhancers fulfill this role. The +9.5 intronic enhancer harbors multiple conserved cis-elements, and germline mutations of these cis-elements are pathogenic in humans. Since mechanisms underlying how GATA2 enhancer disease mutations impact hematopoiesis and pathology are unclear, we generated mouse models of the enhancer mutations. While a multi-motif mutant was embryonic lethal, a single-nucleotide Ets motif mutant was viable, and steady-state hematopoiesis was normal. However, the Ets motif mutation abrogated stem/progenitor cell regeneration following stress. These results reveal a new mechanism in human genetics in which a disease predisposition mutation inactivates enhancer regenerative activity, while sparing developmental activity. Mutational sensitization to stress that instigates hematopoietic failure constitutes a paradigm for GATA-2-deficiency syndrome and other contexts of GATA-2-dependent pathogenesis.
Alexandra A. Soukup, Ye Zheng, Charu Mehta, Jun Wu, Peng Liu, Miao Cao, Inga Hofmann, Yun Zhou, Jing Zhang, Kirby D. Johnson, Kyunghee Choi, Sunduz Keles, Emery H. Bresnick
Targeted therapy with small molecules directed at essential survival pathways in leukemia represents a major advance, including the phosphatidylinositol-3′-kinase (PI3K) p110δ inhibitor idelalisib. Here, we found that genetic inactivation of p110δ (p110δD910A/D910A) in the Eμ-TCL1 murine chronic lymphocytic leukemia (CLL) model impaired B cell receptor signaling and B cell migration, and significantly delayed leukemia pathogenesis. Regardless of TCL1 expression, p110δ inactivation led to rectal prolapse in mice resembling autoimmune colitis in patients receiving idelalisib. Moreover, we showed that p110δ inactivation in the microenvironment protected against CLL and acute myeloid leukemia. After receiving higher numbers of TCL1 leukemia cells, half of p110δD910A/D910A mice spontaneously recovered from high disease burden and resisted leukemia rechallenge. Despite disease resistance, p110δD910A/D910A mice exhibited compromised CD4+ and CD8+ T cell response, and depletion of CD4+ or CD8+ T cells restored leukemia. Interestingly, p110δD910A/D910A mice showed significantly impaired Treg expansion that associated with disease clearance. Reconstitution of p110δD910A/D910A mice with p110δWT/WT Tregs reversed leukemia resistance. Our findings suggest that p110δ inhibitors may have direct antileukemic and indirect immune-activating effects, further supporting that p110δ blockade may have a broader immune-modulatory role in types of leukemia that are not sensitive to p110δ inhibition.
Shuai Dong, Bonnie K. Harrington, Eileen Y. Hu, Joseph T. Greene, Amy M. Lehman, Minh Tran, Ronni L. Wasmuth, Meixiao Long, Natarajan Muthusamy, Jennifer R. Brown, Amy J. Johnson, John C. Byrd
Current thalassemia gene therapy protocols require the collection of hematopoietic stem/progenitor cells (HSPCs), in vitro culture, lentivirus vector transduction, and retransplantation into myelo-ablated patients. Because of cost and technical complexity, it is unlikely that such protocols will be applicable in developing countries where the greatest demand for a beta-thalassemia therapy lies. We have developed a simple in vivo HSPC gene therapy approach that involved HSPC mobilization and an intravenous injection of integrating HDAd5/35++ vectors. Transduced HSPCs homed back to the bone marrow where they persisted long-term. HDAd5/35++ vectors for in vivo gene therapy of thalassemia had a unique capsid that targeted primitive HSPCs through human CD46, a relatively safe SB100X transposase-based integration machinery, a micro-LCR driven gamma-globin gene and, a MGMT(P140K) system that allowed for increasing the therapeutic effect by short-term treatment with low-dose O6BG/BCNU. We showed in “healthy” human CD46 transgenic mice and in a mouse model of thalassemia intermedia that our in vivo approach resulted in stable gamma-globin expression in the majority of circulating red blood cells. The high marking frequency was maintained in secondary recipients. In the thalassemia model, a near complete phenotypic correction was achieved. The treatment was well tolerated. This cost-efficient and “portable” approach could permit a broader clinical application of thalassemia gene therapy.
Hongjie Wang, Aphrodite Georgakopoulou, Nikoletta Psatha, Chang Li, Chrysi Capsali, Himanshu Bhusan Samal, Achilles Anagnostopoulos, Anja Ehrhardt, Zsuzsanna Izsvák, Thalia Papayannopoulou, Evangelia Yannaki, André Lieber
Arteriolar endothelial cell–expressed (EC-expressed) α-globin binds endothelial NOS (eNOS) and degrades its enzymatic product, NO, via dioxygenation, thereby lessening the vasodilatory effects of NO on nearby vascular smooth muscle. Although this reaction potentially affects vascular physiology, the mechanisms that regulate α-globin expression and dioxygenase activity in ECs are unknown. Without β-globin, α-globin is unstable and cytotoxic, particularly in its oxidized form, which is generated by dioxygenation and recycled via endogenous reductases. We show that the molecular chaperone α-hemoglobin–stabilizing protein (AHSP) promotes arteriolar α-globin expression in vivo and facilitates its reduction by eNOS. In Ahsp−/− mice, EC α-globin was decreased by 70%. Ahsp−/− and Hba1−/− mice exhibited similar evidence of increased vascular NO signaling, including arteriolar dilation, blunted α1-adrenergic vasoconstriction, and reduced blood pressure. Purified α-globin bound eNOS or AHSP, but not both together. In ECs in culture, eNOS or AHSP enhanced α-globin expression posttranscriptionally. However, only AHSP prevented oxidized α-globin precipitation in solution. Finally, eNOS reduced AHSP-bound α-globin approximately 6-fold faster than did the major erythrocyte hemoglobin reductases (cytochrome B5 reductase plus cytochrome B5). Our data support a model whereby redox-sensitive shuttling of EC α-globin between AHSP and eNOS regulates EC NO degradation and vascular tone.
Christophe Lechauve, Joshua T. Butcher, Abdullah Freiwan, Lauren A. Biwer, Julia M. Keith, Miranda E. Good, Hans Ackerman, Heather S. Tillman, Laurent Kiger, Brant E. Isakson, Mitchell J. Weiss
Concordant activation of MYC and BCL-2 oncoproteins in double-hit lymphoma (DHL) results in aggressive disease that is refractory to treatment. By integrating activity-based proteomic profiling and drug screens, polo-like kinase-1 (PLK1) was identified as an essential regulator of the MYC-dependent kinome in DHL. Notably, PLK1 was expressed at high levels in DHL, correlated with MYC expression and connoted poor outcome. Further, PLK1 signaling augmented MYC protein stability and, in turn, MYC directly induced PLK1 transcription, establishing a feed-forward MYC-PLK1 circuit in DHL. Finally, inhibition of PLK1 triggered degradation of MYC and of the anti-apoptotic protein MCL1, and PLK1 inhibitors showed synergy with BCL-2 antagonists in blocking DHL cell growth, survival and tumorigenicity, supporting clinical targeting of PLK1 in DHL.
Yuan Ren, Chengfeng Bi, Xiaohong Zhao, Tint Lwin, Cheng Wang, Ji Yuan, Ariosto S. Silva, Bijal D. Shah, Bin Fang, Tao Li, John M. Koomen, Huijuan Jiang, Julio C. Chavez, Lan Pham, Praneeth R. Sudalagunta, Lixin Wan, Xuefeng Wang, William S. Dalton, Lynn C. Moscinski, Kenneth H. Shain, Julie Vose, John L. Cleveland, Eduardo M. Sotomayor, Kai Fu, Jianguo Tao
MASTL, a Ser/Thr kinase that inhibits PP2A-B55 complexes during mitosis, is mutated in autosomal dominant thrombocytopenia. However, the connections between the cell cycle machinery and this human disease remain unexplored. We report here that, whereas Mastl ablation in megakaryocytes prevented proper maturation of these cells, mice carrying the thrombocytopenia-associated mutation developed thrombocytopenia as a consequence of aberrant activation and survival of platelets. Activation of mutant platelets was characterized by hyper-stabilized pseudopods mimicking the effect of PP2A inhibition and actin polymerization defects. These aberrations were accompanied by abnormal hyper-phosphorylation of multiple components of the actin cytoskeleton and were rescued both in vitro and in vivo by inhibiting upstream kinases such as PKA, PKC, or AMPK. These data reveal an unexpected role of Mastl in actin cytoskeleton dynamics in postmitotic cells, and suggest that the thrombocytopenia-associated mutation in MASTL is a pathogenic dominant mutation that mimics decreased PP2A activity resulting in altered phosphorylation of cytoskeletal regulatory pathways.
Begoña Hurtado, Marianna Trakala, Pilar Ximénez-Embún, Aicha El Bakkali, David Partida, Belén Sanz-Castillo, Mónica Álvarez-Fernández, María Maroto, Ruth Sánchez-Martínez, Lola Martínez, Javier Muñoz, Pablo García de Frutos, Marcos Malumbres
Acute Myeloid Leukemia and Myelodysplastic Syndromes are associated with disease-initiating stem cells that are not eliminated by conventional therapies. Transcriptomic analysis of stem and progenitor populations in MDS and AML demonstrated overexpression of STAT3 that was validated in an independent cohort. STAT3 overexpression was predictive of a shorter survival and worse clinical features in a large MDS cohort. High STAT3 expression signature in MDS CD34+ cells was similar to known pre-leukemic gene signatures. Functionally, STAT3 inhibition by a clinical, antisense oligonucleotide, AZD9150, led to reduced viability and increased apoptosis in leukemic cell lines. AZD9150 was rapidly incorporated by primary MDS/AML stem and progenitor cells and led to increased hematopoietic differentiation. STAT3 knockdown also impaired leukemic growth in vivo and led to decreased expression of MCL1 and other oncogenic genes in malignant cells. These studies demonstrate that STAT3 is an adverse prognostic factor in MDS/AML and provide a pre-clinical rationale for studies using AZD9150 in these diseases.
Aditi Shastri, Gaurav Choudhary, Margarida Teixeira, Shanisha Gordon-Mitchell, Nandini Ramachandra, Lumie Bernard, Sanchari Bhattacharyya, Robert Lopez, Kith Pradhan, Orsolya Giricz, Goutham Ravipati, Li-Fan Wong, Sally Cole, Tushar D. Bhagat, Jonathan Feld, Yosman Dhar, Matthias Bartenstein, Victor J. Thiruthuvanathan, Amittha Wickrema, B. Hilda Ye, David A. Frank, Andrea Pellagatti, Jacqueline Boultwood, Tianyuan Zhou, Youngsoo Kim, A. Robert MacLeod, Pearlie K. Epling-Burnette, Minwei Ye, Patricia McCoon, Richard Woessner, Ulrich Steidl, Britta Will, Amit K. Verma
ASXL1 is frequently mutated in myeloid malignancies and is known to co-occur with other gene mutations. However, the molecular mechanisms underlying the leukemogenesis associated with ASXL1 and cooperating mutations remain to be elucidated. Here we report that Asxl1 loss cooperated with haploinsufficiency of Nf1, a negative regulator of the RAS signaling pathway, to accelerate the development of myeloid leukemia in mice. Loss of Asxl1 and Nf1 in hematopoietic stem and progenitor cells resulted in a gain-of-function transcriptional activation of multiple pathways critical for leukemogenesis, such as MYC, NRAS, and BRD4. The hyperactive MYC and BRD4 transcription programs were correlated with elevated H3K4 tri-methylation at the promoter regions of genes involving these pathways. Furthermore, pharmacological inhibition of both MAPK pathway and BET bromodomain prevented leukemia initiation and inhibited disease progression in Asxl1Δ/Δ;Nf1Δ/Δ mice. Concomitant mutations of ASXL1 and RAS pathway genes were associated with aggressive progression of myeloid malignancies in patients. This study sheds light on the understanding of the cooperative effect between epigenetic alterations and signaling pathways in accelerating the progression of myeloid malignancies and provides a rational therapeutic strategy for the treatment of myeloid malignancies with ASXL1 and RAS pathway gene mutations.
Peng Zhang, Fuhong He, Jie Bai, Shohei Yamamoto, Shi Chen, Lin Zhang, Mengyao Sheng, Lei Zhang, Ying Guo, Na Man, Hui Yang, Suyun Wang, Tao Cheng, Stephen D. Nimer, Yuan Zhou, Mingjiang Xu, Qian-Fei Wang, Feng-Chun Yang
Quantitative abnormalities of the von Willebrand factor–factor VIII (VWF-FVIII) complex associate with inherited bleeding or thrombotic disorders. Receptor-mediated interactions between plasma VWF-FVIII and phagocytic or immune cells can influence their hemostatic and immunogenic activities. Genetic association studies have demonstrated that variants in the STAB2 gene, which encodes the scavenger receptor stabilin-2, associate with plasma levels of VWF-FVIII. However, the mechanistic basis and pathophysiological consequences of this association are unknown. We have demonstrated that stabilin-2–expressing cells bind and internalize human VWF and FVIII in a VWF-dependent manner, and stabilin-2–deficient mice displayed prolonged human VWF-FVIII half-life compared with controls. The stabilin-2 variant p.E2377K significantly decreased stabilin-2 expression and impaired VWF endocytosis in a heterologous expression system, and common STAB2 variants associated with plasma VWF levels in type 1 von Willebrand disease patients. STAB2-deficient mice displayed a decreased immunogenic response to human VWF-FVIII complex, while coinfusion of human VWF-FVIII with the stabilin-2 ligand hyaluronic acid attenuated the immune response to exogenous FVIII. Collectively, these data suggest that stabilin-2 functions as both a clearance and an immunoregulatory receptor for VWF-FVIII, making stabilin-2 a novel molecular target for modification of the half-life of VWF-FVIII and the immune response to VWF-FVIII concentrates.
Laura L. Swystun, Jesse D. Lai, Colleen Notley, Ilinca Georgescu, A. Simonne Paine, Jeff Mewburn, Kate Nesbitt, Kai Schledzewski, Cyrill Géraud, Julia Kzhyshkowska, Sergij Goerdt, Wilma Hopman, Robert R. Montgomery, Paula D. James, David Lillicrap
Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) is a new pathological entity with poor outcomes in T cell ALL (T-ALL) that is characterized by a high incidence of loss-of-function mutations in polycomb repressive complex 2 (PRC2) genes. We generated a mouse model of ETP-ALL by deleting Ezh2, one of the PRC2 genes, in p53-null hematopoietic cells. The loss of Ezh2 in p53-null hematopoietic cells impeded the differentiation of ETPs and eventually induced ETP-ALL–like disease in mice, indicating that PRC2 functions as a bona fide tumor suppressor in ETPs. A large portion of PRC2 target genes acquired DNA hypermethylation of their promoters following reductions in H3K27me3 levels upon the loss of Ezh2, which included pivotal T cell differentiation–regulating genes. The reactivation of a set of regulators by a DNA-demethylating agent, but not the transduction of single regulator genes, effectively induced the differentiation of ETP-ALL cells. Thus, PRC2 protects key T cell developmental regulators from DNA hypermethylation in order to keep them primed for activation upon subsequent differentiation phases, while its insufficiency predisposes ETPs to leukemic transformation. These results revealed a previously unrecognized epigenetic switch in response to PRC2 dysfunction and provide the basis for specific rational epigenetic therapy for ETP-ALL with PRC2 insufficiency.
Changshan Wang, Motohiko Oshima, Daisuke Sato, Hirotaka Matsui, Sho Kubota, Kazumasa Aoyama, Yaeko Nakajima-Takagi, Shuhei Koide, Jun Matsubayashi, Makiko Mochizuki-Kashio, Takako Nakano-Yokomizo, Jie Bai, Toshitaka Nagao, Akinori Kanai, Atsushi Iwama, Goro Sashida