Anemia in β-thalassemia is related to ineffective erythropoiesis and reduced red cell survival. Excess free heme and accumulation of unpaired α-globin chains impose substantial oxidative stress on β-thalassemic erythroblasts and erythrocytes, impacting cell metabolism. We hypothesized that increased pyruvate kinase activity induced by mitapivat (AG-348) in the Hbbth3/+ mouse model for β-thalassemia would reduce chronic hemolysis and ineffective erythropoiesis through stimulation of red cell glycolytic metabolism. Oral mitapivat administration ameliorated ineffective erythropoiesis and anemia in Hbbth3/+ mice. Increased ATP, reduced reactive oxygen species production, and reduced markers of mitochondrial dysfunction associated with improved mitochondrial clearance suggested enhanced metabolism following mitapivat administration in β-thalassemia. The amelioration of responsiveness to erythropoietin resulted in reduced soluble erythroferrone, increased liver Hamp expression, and diminished liver iron overload. Mitapivat reduced duodenal Dmt1 expression potentially by activating the pyruvate kinase M2HIF2α axis, representing a mechanism additional to Hamp in controlling iron absorption and preventing β-thalassemia–related liver iron overload. In ex vivo studies on erythroid precursors from patients with β-thalassemia, mitapivat enhanced erythropoiesis, promoted erythroid maturation, and decreased apoptosis. Overall, pyruvate kinase activation as a treatment modality for β-thalassemia in preclinical model systems had multiple beneficial effects in the erythropoietic compartment and beyond, providing a strong scientific basis for further clinical trials.
Alessandro Matte, Enrica Federti, Charles Kung, Penelope A. Kosinski, Rohini Narayanaswamy, Roberta Russo, Giorgia Federico, Francesca Carlomagno, Maria Andrea Desbats, Leonardo Salviati, Christophe Leboeuf, Maria Teresa Valenti, Francesco Turrini, Anne Janin, Shaoxia Yu, Elisabetta Beneduce, Sebastien Ronseaux, Iana Iatcenko, Lenny Dang, Tomas Ganz, Chun-Ling Jung, Achille Iolascon, Carlo Brugnara, Lucia De Franceschi
Inhibitors to factor VIII (FVIII) remain the most challenging complication of FVIII protein replacement therapy in hemophilia A (HA). Understanding the mechanisms that guide FVIII-specific B cell development could help identify therapeutic targets. The B cell activating factor (BAFF) cytokine family is a key regulator of B cell differentiation in normal homeostasis and immune disorders. Thus, we used patient samples and mouse models to investigate the potential role of BAFF in modulating FVIII inhibitors. BAFF levels were elevated in pediatric and adult HA inhibitor patients and decreased to levels similar to non-inhibitor controls after successful immune tolerance induction (ITI). Moreover, elevations in BAFF levels were seen in patients who failed to achieve FVIII tolerance with anti-CD20 mediated B-cell depletion. In naïve HA mice, prophylactic anti-BAFF therapy prior to FVIII immunization prevented inhibitors and this tolerance was maintained despite FVIII exposure after immune reconstitution. In preimmunized HA mice, combination therapy with anti-CD20 and anti-BAFF antibodies dramatically reduced FVIII inhibitors via inhibition of FVIII-specific plasma cells. Our data suggest that BAFF may regulate the generation and maintenance of FVIII inhibitors and/or anti-FVIII B cells. Finally, anti-CD20/anti-BAFF combination therapy may be clinically useful for ITI.
Bhavya S. Doshi, Jyoti Rana, Giancarlo Castaman, Mostafa A. Shaheen, Radoslaw Kaczmarek, John S. S. Butterfield, Shannon L. Meeks, Cindy Leissinger, Moanaro Biswas, Valder R. Arruda
In order to sustain proficient life-long hematopoiesis, hematopoietic stem cells (HSCs) must possess robust mechanisms to preserve their quiescence and genome integrity. DNA-damaging stress can perturb HSC homeostasis by affecting their survival, self-renewal and differentiation. Ablation of the kinase ATM, a master regulator of the DNA damage response, impairs HSC fitness. Paradoxically, we show here that loss of a single allele of Atm enhances HSC functionality in mice. To explain this observation, we explored a possible link between ATM and the tumor suppressor PTEN, which also regulates HSC function. We generated and analyzed a knock-in mouse line (PtenS398A/S398A), in which PTEN cannot be phosphorylated by ATM. Similar to Atm+/-, PtenS398A/S398A HSCs have enhanced hematopoietic reconstitution ability, accompanied by resistance to apoptosis induced by genotoxic stress. Single-cell transcriptomic analyses and functional assays revealed that dormant PtenS398A/S398A HSCs aberrantly tolerate elevated mitochondrial activity and the accumulation of reactive oxygen species, which are normally associated with HSC priming for self-renewal or differentiation. Our results unveil a molecular connection between ATM and PTEN, which couples the response to genotoxic stress and dormancy in HSC.
Jerome Fortin, Christian Bassi, Parameswaran Ramachandran, Wanda Y. Li, Ruxiao Tian, Ida Zarrabi, Graham Hill, Bryan E. Snow, Jillian Haight, Chantal Tobin, Kelsey Hodgson, Andrew Wakeham, Vuk Stambolic, Tak W. Mak
Bone marrow (BM) hematopoietic stem cells (HSCs) become dysfunctional during aging (i.e., they are increased in number but have an overall reduction in long-term repopulation potential and increased myeloid differentiation) compared with young HSCs, suggesting limited use of old donor BM cells for hematopoietic cell transplantation (HCT). BM cells reside in an in vivo hypoxic environment yet are evaluated after collection and processing in ambient air. We detected an increase in the number of both young and aged mouse BM HSCs collected and processed in 3% O2 compared with the number of young BM HSCs collected and processed in ambient air (~21% O2). Aged BM collected and processed under hypoxic conditions demonstrated enhanced engraftment capability during competitive transplantation analysis and contained more functional HSCs as determined by limiting dilution analysis. Importantly, the myeloid-to-lymphoid differentiation ratio of aged BM collected in 3% O2 was similar to that detected in young BM collected in ambient air or hypoxic conditions, consistent with the increased number of common lymphoid progenitors following collection under hypoxia. Enhanced functional activity and differentiation of old BM collected and processed in hypoxia correlated with reduced “stress” associated with ambient air BM collection and suggests that aged BM may be better and more efficiently used for HCT if collected and processed under hypoxia so that it is never exposed to ambient air O2.
Maegan L. Capitano, Safa F. Mohamad, Scott Cooper, Bin Guo, Xinxin Huang, Andrea M. Gunawan, Carol Sampson, James Ropa, Edward F. Srour, Christie M. Orschell, Hal E. Broxmeyer
Autosomal dominant "sterile alpha motif domain containing 9 (Samd9) and Samd9L (Samd9/9L) syndromes" are a large subgroup of currently established inherited bone marrow failure syndromes that include MIRAGE, ataxia pancytopenia, and familial monosomy 7 syndromes. Samd9/9L genes are located in tandem on chromosome 7 and have been known to be the genes responsible for myeloid malignancies associated with monosomy 7. Additionally, as interferon-inducible genes, Samd9/9L are crucial for protection against viruses. Samd9/9L syndromes are caused by gain-of-function mutations and develop into infantile myelodysplastic syndromes associated with monosomy 7 (MDS/-7) at extraordinarily high frequencies. We generated mice expressing Samd9LD764N, which mimic the MIRAGE syndrome presenting with growth retardation, a short life, bone marrow failure, and multi-organ degeneration. In hematopoietic cells, Samd9LD764N downregulates the endocytosis of transferrin and c-Kit resulting in a rare cause of anemia and a low bone marrow reconstitutive potential that ultimately causes MDS/-7. By contrast, in non-hematopoietic cells we tested, Samd9LD764N upregulated the endocytosis of EGFR by Ship2 phosphatase translocation to the cytomembrane and activated lysosomes, resulting in the reduced expression of surface receptors and signaling. Thus Samd9/9L is a downstream regulator of interferon that controls receptor metabolism, with constitutive activation leading to multi-organ dysfunction.
Akiko Nagamachi, Akinori Kanai, Megumi Nakamura, Hiroshi Okuda, Akihiko Yokoyama, Satoru Shinriki, Hirotaka Matsui, Toshiya Inaba
How particular bone marrow niche factors contribute to the leukemogenic activities of leukemia-initiating cells (LICs) remain largely unknown. Here, we showed that ATP levels were markedly increased in the bone marrow niches of mice with acute myeloid leukemia (AML), and LICs preferred to localizing to the endosteal niche with relatively high ATP levels, as indicated by a sensitive ATP indicator. ATP could efficiently induce the influx of ions into LICs in an MLL-AF9-induced murine AML model via the ligand-gated ion channel P2X7. P2x7 deletion led to notably impaired homing and self-renewal capacities of LICs and contributed to an ~5-fold decrease in the number of functional LICs but had no effect on normal hematopoiesis. ATP-P2X7 signaling enhanced the calcium flux-mediated phosphorylation of CREB, which further transactivated the Phgdh expression to maintain serine metabolism and LIC fates. P2X7-knockdown resulted in a markedly extended survival of recipients transplanted with either human AML cell lines or primary leukemia cells. Blockade of ATP-P2X7 signaling could efficiently inhibit leukemogenesis. Here, we provide a unique perspective for understanding how ATP-P2X7 signaling sustains the LIC activities, which may benefit the development of specific strategies for targeting LICs or other types of cancer stem cells
Xiaoxiao He, Jiangbo Wang, Xiaona Yang, Xiuze Zhang, Dan Huang, Xie Li, Yejun Zou, Chiqi Chen, Zhuo Yu, Li Xie, Yaping Zhang, Ligen Liu, Shangang Li, Yuzheng Zhao, Hongfang Shao, Ye Yu, Junke Zheng
Cutaneous T cell lymphoma (CTCL) has a poorly understood etiology and no cure. Using conditional knockout mice, we found that ablation of the genomic organizer Special AT-rich sequence-binding protein-1 (Satb1) caused malignant transformation of mature, skin-homing, Notch-activated CD4+ and CD8+ T-cells into progressively fatal lymphoma. Mechanistically, Satb1 restrained Stat5 phosphorylation and the expression of skin-homing chemokine receptors in mature T-cells. Notably, methyltransferase-dependent epigenetic repression of SATB1 was universally found in human Sézary syndrome, but not in other peripheral T-cell malignancies. H3K27 and H3K9 trimethylation occluded the SATB1 promoter in Sézary cells, while inhibition of SUV39H1/2 methyltransferases (unlike EZH2 inhibition), restored protective SATB1 expression and selectively abrogated the growth of primary Sézary cells more effectively than romidepsin. Therefore, inhibition of methyltransferases that silence SATB1 could address an unmet need for patients with Mycosis fungoides/Sézary syndrome, a set of incurable diseases.
Carly M. Harro, Jairo Perez-Sanz, Tara Lee Costich, Kyle K. Payne, Carmen M. Anadon Galindo, Ricardo A. Chaurio, Subir Biswas, Gunjan Mandal, Kristen E. Rigolizzo, Kimberly B. Sprenger, Jessica A. Mine, Louise Showe, Xiaoqing Yu, Kebin Liu, Paulo C. Rodriguez, Javier Pinilla-Ibarz, Lubomir Sokol, Jose R. Conejo-Garcia
BACKGROUND Interpatient differences in the accumulation of methotrexate’s active polyglutamylated metabolites (MTXPGs) in leukemia cells influence its antileukemic effects.METHODS To identify genomic and epigenomic and patient variables determining the intracellular accumulation of MTXPGs, we measured intracellular MTXPG levels in acute lymphoblastic leukemia (ALL) cells from 388 newly diagnosed patients after in vivo high-dose methotrexate (HDMTX) (1 g/m2) treatment, defined ALL subtypes, and assessed genomic and epigenomic variants influencing folate pathway genes (mRNA, miRNA, copy number alterations [CNAs], SNPs, single nucleotide variants [SNVs], CpG methylation).RESULTS We documented greater than 100-fold differences in MTXPG levels, which influenced its antileukemic effects (P = 4 × 10–5). Three ALL subtypes had lower MTXPG levels (T cell ALL [T-ALL] and B cell ALL [B-ALL] with the TCF3-PBX1 or ETV6-RUNX1 fusions), and 2 subtypes had higher MTXPG levels (hyperdiploid and BCR-ABL like). The folate pathway genes SLC19A1, ABCC1, ABCC4, FPGS, and MTHFD1 significantly influenced intracellular MTXPG levels (P = 2.9 × 10–3 to 3.7 × 10–8). A multivariable model including the ALL subtype (P = 1.1 × 10–14), the SLC19A1/(ABCC1 + ABCC4) transporter ratio (P = 3.6 × 10–4), the MTX infusion time (P = 1.5 × 10–3), FPGS mRNA expression (P = 2.1 × 10–3), and MTX systemic clearance (P = 4.4 × 10–2) explained 42% of the variation in MTXPG accumulation (P = 1.1 × 10–38). Model simulations indicated that a longer infusion time (24 h vs. 4 h) was superior in achieving higher intracellular MTXPG levels across all subtypes if ALL.CONCLUSIONS These findings provide insights into mechanisms underlying interpatient differences in intracellular accumulation of MTXPG in leukemia cells and its antileukemic effectsFUNDING THE National Cancer Institute (NCI) and the Institute of General Medical Sciences of the NIH, the Basque Government Programa Posdoctoral de Perfeccionamiento de Personal Investigador doctor, and the American Lebanese Syrian Associated Charities (ALSAC).
Elixabet Lopez-Lopez, Robert J. Autry, Colton Smith, Wenjian Yang, Steven W. Paugh, John C. Panetta, Kristine R. Crews, Erik J. Bonten, Brandon Smart, Deqing Pei, J. Robert McCorkle, Barthelemy Diouf, Kathryn G. Roberts, Lei Shi, Stanley Pounds, Cheng Cheng, Charles G. Mullighan, Ching-Hon Pui, Mary V. Relling, William E. Evans
Anaplastic large cell lymphoma (ALCL) is a mature T cell neoplasm that often expresses the CD4+ T cell surface marker. It usually harbors the t(2;5) (p23;q35) translocation, leading to the ectopic expression of NPM-ALK, a chimeric tyrosine kinase. We demonstrated that in vitro transduction of normal human CD4+ T lymphocytes with NPM-ALK results in their immortalization and malignant transformation. The tumor cells displayed morphological and immunophenotypical characteristics of primary patient–derived anaplastic large cell lymphomas. Cell growth, proliferation, and survival were strictly dependent on NPM-ALK activity and include activation of the key factors STAT3 and DNMT1 and expression of CD30 (the hallmark of anaplastic large-cell lymphoma). Implantation of NPM-ALK–transformed CD4+ T lymphocytes into immunodeficient mice resulted in the formation of tumors indistinguishable from patients’ anaplastic large cell lymphomas. Integration of “Omic” data revealed that NPM-ALK–transformed CD4+ T lymphocytes and primary NPM-ALK+ ALCL biopsies share similarities with early T cell precursors. Of note, these NPM-ALK+ lymphoma cells overexpress stem cell regulators (OCT4, SOX2, and NANOG) and HIF2A, which is known to affect hematopoietic precursor differentiation and NPM-ALK+ cell growth. Altogether, for the first time our findings suggest that NPM-ALK could restore progenitor-like features in mature CD30+ peripheral CD4+ T cells, in keeping with a thymic progenitor-like pattern.
Annabelle Congras, Coralie Hoareau-Aveilla, Nina Caillet, Marie Tosolini, Patrick Villarese, Agata Cieslak, Laura Rodriguez, Vahid Asnafi, Elisabeth Macintyre, Gerda Egger, Pierre Brousset, Laurence Lamant, Fabienne Meggetto
Small extracellular vesicles (SEVs) are functional messengers of certain cellular niches to permit non-contact cell communications. Whether niche-specific SEVs fulfill this role in cancer is unclear. Here, we used seven cell-type specific mouse Cre lines to conditionally knockout Vps33b in Cdh5+ or Tie2+ endothelial cells (ECs), Lepr+ bone marrow perivascular cells, Osx+ osteo-progenitor cells (OPCs), Pf4+ megakaryocytes and Tcf21+ spleen stromal cells. We then examined the effects of reduced SEV secretion on progression of MLL-AF9 induced acute myeloid leukemia (AML) as well as normal hematopoiesis. Blocking SEV secretion from ECs, but not perivascular cells, megakaryocytes or spleen stromal cells, markedly delayed the leukemia progression. Notably, reducing SEV production from ECs had no effect on normal hematopoiesis. Protein analysis showed that EC-derived SEVs contained a high level of ANGPTL2, which accelerated leukemia progression via binding to LILRB2 receptor. Moreover, ANGPTL2-SEVs released from ECs were governed by VPS33B. Importantly, ANGPTL2-SEVs were also required for primary human AML cell maintenance. These findings demonstrate a role of niche-specific SEVs in cancer development and suggest that targeting ANGPTL2-SEVs from ECs might be a potential strategy to interfere certain types of AML.
Dan Huang, Guohuan Sun, Xiaoxin Hao, Xiaoxiao He, Zhaofeng Zheng, Chiqi Chen, Zhuo Yu, Li Xie, Shihui Ma, Ligen Liu, Bo O. Zhou, Hui Cheng, Junke Zheng, Tao Cheng