SAMD9 and SAMD9L germline mutations have recently emerged as a new class of predispositions to pediatric myeloid neoplasms. Patients commonly have impaired hematopoiesis, hypocellular marrows, and a greater risk of developing clonal chromosome 7 deletions leading to MDS and AML. We recently demonstrated that expressing SAMD9 or SAMD9L mutations in hematopoietic cells suppresses their proliferation and induces cell death. Here we generated a mouse model that conditionally expresses mutant Samd9l to assess the in vivo impact on hematopoiesis. Using a range of in vivo and ex vivo assays, we showed that cells with heterozygous Samd9l mutations have impaired stemness relative to wild-type counterparts, which was exacerbated by inflammatory stimuli, and ultimately led to bone marrow hypocellularity. Genomic and phenotypic analyses recapitulated many of the hematopoietic cellular phenotypes observed in patients with SAMD9 or SAMD9L mutations, including lymphopenia, and pinpointed TGF-β as a potential targetable pathway. Further, we observed non-random genetic deletion of the mutant Samd9l locus on mouse chromosome 6, mimicking chromosome 7 deletions observed in patients. Collectively, our study has enhanced our understanding of mutant Samd9l hematopoietic phenotypes, emphasized the synergistic role of inflammation in exaggerating the associated hematopoietic defects, and provided insights into potential therapeutic options for patients.
Sherif Abdelhamed, Melvin E. Thomas III, Tamara Westover, Masayuki Umeda, Emily Xiong, Chandra Rolle, Michael P. Walsh, Huiyun Wu, Jason R. Schwartz, Virginia Valentine, Marcus Valentine, Stanley Pounds, Jing Ma, Laura J. Janke, Jeffery M. Klco
In chronic myeloid leukemia (CML), combination therapies with tyrosine kinase inhibitors (TKIs) aim to improve the achievement of deep molecular remission that would allow therapy discontinuation. IFN-α is one promising candidate, as it has long-lasting effects on both malignant and immune cells. In connection with a multicenter clinical trial combining dasatinib with IFN-α in 40 patients with chronic-phase CML (NordCML007, NCT01725204), we performed immune monitoring with single-cell RNA and T cell receptor (TCR) sequencing (n = 4, 12 samples), bulk TCRβ sequencing (n = 13, 26 samples), flow cytometry (n = 40, 106 samples), cytokine analyses (n = 17, 80 samples), and ex vivo functional studies (n = 39, 80 samples). Dasatinib drove the immune repertoire toward terminally differentiated NK and CD8+ T cells with dampened functional capabilities. Patients with dasatinib-associated pleural effusions had increased numbers of CD8+ recently activated effector memory T (Temra) cells. In vitro, dasatinib prevented CD3-induced cell death by blocking TCR signaling. The addition of IFN-α reversed the terminally differentiated phenotypes and increased the number of costimulatory intercellular interactions and the number of unique putative epitope-specific TCR clusters. In vitro IFN-α had costimulatory effects on TCR signaling. Our work supports the combination of IFN-α with TKI therapy, as IFN-α broadens the immune repertoire and restores immunological function.
Jani Huuhtanen, Mette Ilander, Bhagwan Yadav, Olli M.J. Dufva, Hanna Lähteenmäki, Tiina Kasanen, Jay Klievink, Ulla Olsson-Strömberg, Jesper Stentoft, Johan Richter, Perttu Koskenvesa, Martin Höglund, Stina Söderlund, Arta Dreimane, Kimmo Porkka, Tobias Gedde-Dahl, Björn T. Gjertsen, Leif Stenke, Kristina Myhr-Eriksson, Berit Markevärn, Anna Lübking, Andreja Dimitrijevic, Lene Udby, Ole Weis Bjerrum, Henrik Hjorth-Hansen, Satu Mustjoki
Infantile (fetal and neonatal) megakaryocytes have a distinct phenotype consisting of hyperproliferation, limited morphogenesis, and low platelet production capacity. These properties contribute to clinical problems that include thrombocytopenia in neonates, delayed platelet engraftment in recipients of cord blood stem cell transplants, and inefficient ex vivo platelet production from pluripotent stem cell-derived megakaryocytes. The infantile phenotype results from deficiency of the actin-regulated coactivator, MKL1, which programs cytoskeletal changes driving morphogenesis. As a strategy to complement this molecular defect, we screened pathways with potential to affect MKL1 function and found that Dyrk1a kinase inhibition dramatically enhanced megakaryocyte morphogenesis in vitro and in vivo. Dyrk1 inhibitors rescued enlargement, polyploidization, and thrombopoiesis in human neonatal megakaryocytes. Megakaryocytes derived from induced pluripotent stem cells responded in a similar manner. Progenitors undergoing Dyrk1 inhibition demonstrated filamentous actin assembly, MKL1 nuclear translocation, and modulation of MKL1 target genes. Loss of function studies confirmed MKL1 involvement in this morphogenetic pathway. Ablim2, a stabilizer of filamentous actin, increased with Dyrk1 inhibition, and Ablim2 knockdown abrogated the actin, MKL1, and morphogenetic responses to Dyrk1 inhibition. These results thus delineate a pharmacologically tractable morphogenetic pathway whose manipulation may alleviate clinical problems associated with the limited thrombopoietic capacity of infantile megakaryocytes.
Kamaleldin E. Elagib, Ashton Brock, Cara M. Clementelli, Gohar Mosoyan, Lorrie L. Delehanty, Ranjit K. Sahu, Alexandra Pacheco-Benichou, Corinne Fruit, Thierry Besson, Stephan W. Morris, Koji Eto, Chintan Jobaliya, Deborah L. French, Paul Gadue, Sandeep Singh, Xinrui Shi, Fujun Qin, Robert Cornelison, Hui Li, Camelia Iancu-Rubin, Adam N. Goldfarb
Myelodysplastic syndromes (MDS) are age-related myeloid neoplasms with increased risks of progression to acute myeloid leukemia (AML). The mechanisms of MDS to AML transformation are poorly understood, especially in relation to the aging microenvironment. We previously established a mDia1/miR-146a double knockout (DKO) mouse model phenocopying MDS. These mice develop age-related pancytopenia with over-secretion of pro-inflammatory cytokines. Here, we found that most of the DKO mice underwent leukemic transformation at 12-14 months of age. These mice showed myeloblast replacement of a fibrotic bone marrow and widespread leukemic infiltration. Strikingly, depletion of IL-6 in these mice largely rescued the leukemic transformation and markedly extended the survival. Single cell RNA sequencing analyses revealed that DKO leukemic mice had increased monocytic blasts that were reduced with IL-6 knockout. We further revealed that the levels of surface and soluble IL-6 receptor (IL-6R) in the bone marrow were significantly increased in high risk MDS patients. Similarly, IL-6R was also highly expressed in older DKO mice. Blocking of IL-6 signaling significantly ameliorated AML progression in the DKO model and clonogenicity of CD34 positive cells from MDS patients. Our study establishes a mouse model of age-related MDS to AML progression and indicates the clinical significance of targeting IL-6 signaling in treating high risk MDS.
Yang Mei, Kehan Ren, Yijie Liu, Annabel Ma, Zongjun Xia, Xu Han, Ermin Li, Hamza Tariq, Haiyan Bao, Xinshu Xie, Cheng Zou, Dingxiao Zhang, Zhaofeng Li, Lili Dong, Amit Verma, Xinyan Lu, Yasmin Abaza, Jessica K. Altman, Madina Sukhanova, Jing Yang, Peng Ji
Osteolytic bone disease is a hallmark of multiple myeloma (MM). A significant fraction (~20%) of MM patients do not develop osteolytic lesions (OL). The molecular basis for the absence of bone disease in MM is not understood. We combined PET-CT and gene expression profiling (GEP) of purified bone marrow (BM) CD138+ MM cells from 512 newly diagnosed MM patients to reveal that elevated expression of cystatin M/E (CST6) was significantly associated with the absence of OL in MM. An enzyme-linked immunosorbent assay revealed a strong correlation between CST6 levels in BM serum/plasma and CST6 mRNA expression. Both recombinant CST6 protein and BM serum from patients with high CST6 significantly inhibited the activity of the osteoclast-specific protease cathepsin K, and blocked osteoclast differentiation and function. Recombinant CST6 inhibited bone destruction in ex vivo and in vivo myeloma models. Single cell RNA-sequencing identified that CST6 attenuates polarization of monocytes to osteoclast precursors. Furthermore, CST6 protein blocks osteoclast differentiation by suppressing cathepsin-mediated cleavage of NF-κB/p100 and TRAF3 following RANKL stimulation. Secretion by MM cells of CST6, an inhibitor of osteoclast differentiation and function, suppresses osteolytic bone disease in MM and probably other diseases associated with osteoclast-mediated bone loss.
Dongzheng Gai, Jin-Ran Chen, James P. Stewart, Intawat Nookaew, Hasem Habelhah, Cody Ashby, Fumou Sun, Yan Cheng, Can Li, Hongwei Xu, Bailu Peng, Tarun K. Garg, Carolina Schinke, Sharmilan Thanendrarajan, Maurizio Zangari, Fangping Chen, Bart Barlogie, Frits van Rhee, Guido Tricot, John D. Shaughnessy Jr, Fenghuang Zhan
BACKGROUND. Herpes simplex virus lymphadenitis (HSVL) is an unusual presentation of HSV reactivation in chronic lymphocytic leukemia (CLL) patients characterized by systemic symptoms and no herpetic lesions. The immune responses during HSVL have not been studied. METHODS. Peripheral blood and lymph node samples of a patient with HSVL were obtained. HSV-2 viral load, antibody levels, B and T cell responses, cytokine levels, and tumor burden were measured. RESULTS. This patient showed HSV-2 viremia for at least 6 weeks. During this period, she had a robust HSV-specific antibody response with neutralizing and antibody-dependent cellular phagocytosis activity. Activated (HLA-DR+, CD38+) CD4+ and CD8+ T cells increased 18-fold and HSV-specific CD8+ T cells were detected in the blood at higher numbers. HSV-specific B and T cell responses in the lymph node were also detected. Markedly elevated levels of pro-inflammatory cytokines in the blood were also observed. Surprisingly, a sustained decrease in CLL tumor burden without CLL-directed therapy was observed with this and also a prior episode of HSVL. CONCLUSION. HSVL should be considered as part of the differential diagnosis in CLL patients who present with signs and symptoms of aggressive lymphoma transformation. An interesting finding was the sustained tumor control after 2 episodes of HSVL in this patient. This tumor burden reduction may be due to the HSV-specific response serving as an adjuvant for activating tumor-specific or bystander T cells. Studies in additional CLL patients are needed to confirm and extend these findings. FUNDING. National Institutes of Health and Winship Cancer Institute.
Andres Chang, Anton M. Sholukh, Andreas Wieland, David L. Jaye, Mary Carrington, Meei-Li Huang, Hong Xie, Keith R. Jerome, Pavitra Roychoudhury, Alexander L. Greninger, Jean L. Koff, Jonathon B. Cohen, David M. Koelle, Lawrence Corey, Christopher R. Flowers, Rafi Ahmed
Katsuyoshi Takata, Christian Steidl
Defining mechanism(s) that maintain tissue stem quiescence is important for improving tissue regeneration, cell therapies, aging, and cancer. We report here that genetic ablation of Id2 in adult hematopoietic stem cells (HSCs) promotes increased HSC activation and differentiation, which results in HSC exhaustion and bone marrow failure over time. Id2Δ/Δ HSCs showed increased cycling, ROS production, mitochondrial activation, ATP production, and DNA damage compared with Id2+/+ HSCs, supporting the conclusion that Id2Δ/Δ HSCs are less quiescent. Mechanistically, HIF-1α expression was decreased in Id2Δ/Δ HSCs, and stabilization of HIF-1α in Id2Δ/Δ HSCs restored HSC quiescence and rescued HSC exhaustion. Inhibitor of DNA binding 2 (ID2) promoted HIF-1α expression by binding to the von Hippel-Lindau (VHL) protein and interfering with proteasomal degradation of HIF-1α. HIF-1α promoted Id2 expression and enforced a positive feedback loop between ID2 and HIF-1α to maintain HSC quiescence. Thus, sustained ID2 expression could protect HSCs during stress and improve HSC expansion for gene editing and cell therapies.
Brad L. Jakubison, Tanmoy Sarkar, Kristbjorn O. Gudmundsson, Shweta Singh, Lei Sun, Holly M. Morris, Kimberly D. Klarmann, Jonathan R. Keller
Targeted protein degradation is a rapidly advancing and expanding therapeutic approach. Drugs that degrade GSPT1 via the CRL4CRBN ubiquitin ligase are a new class of cancer therapy in active clinical development with evidence of activity against acute myeloid leukemia in early phase trials. However, other than activation of the integrated stress response, the downstream effects of GSPT1 degradation leading to cell death are largely undefined, and no murine models are available to study these agents. We identified the domains of GSPT1 essential for cell survival and show that GSPT1 degradation leads to impaired translation termination, activation of the integrated stress response pathway, and TP53-independent cell death. CRISPR-Cas9 screens implicated decreased translation initiation as protective to GSPT1 degradation, suggesting that cells with higher levels of translation are more susceptible to GSPT1 degradation. We defined two Crbn amino acids that prevent Gspt1 degradation in mice, generated a knock-in mouse with alteration of these residues, and demonstrated the efficacy of GSPT1-degrading drugs in vivo with relative sparing of numbers and function of long-term hematopoietic stem cells. Our results provide a mechanistic basis for the use of GSPT1 degraders for the treatment of cancer, including TP53-mutant AML.
Rob S. Sellar, Adam S. Sperling, Mikołaj Słabicki, Jessica A. Gasser, Marie E. McConkey, Katherine A. Donovan, Nada Mageed, Dylan N. Adams, Charles Zou, Peter G. Miller, Ravi Kumar Dutta, Steffen Boettcher, Amy E. Lin, Brittany E. Sandoval, Vanessa A. Quevedo Barrios, Veronica Shkolnik, Jonas Koeppel, Elizabeth K. Henderson, Emma C. Fink, Lu Yang, Anthony K.N. Chan, Sheela Pangeni Pokharel, Erik J. Bergstrom, Rajan Burt, Namrata D. Udeshi, Steven A. Carr, Eric S. Fischer, Chun-Wei Chen, Benjamin L. Ebert
The crosstalk between the BM microenvironment (niche) and hematopoietic stem cells (HSCs) is critical for HSC regeneration. Here, we show that in mice, deletion of the Fanconi anemia (FA) genes Fanca and Fancc dampened HSC regeneration through direct effects on HSCs and indirect effects on BM niche cells. FA HSCs showed persistent upregulation of the Wnt target Prox1 in response to total body irradiation (TBI). Accordingly, lineage-specific deletion of Prox1 improved long-term repopulation of the irradiated FA HSCs. Forced expression of Prox1 in WT HSCs mimicked the defective repopulation phenotype of FA HSCs. WT mice but not FA mice showed significant induction by TBI of BM stromal Wnt5a protein. Mechanistically, FA proteins regulated stromal Wnt5a expression, possibly through modulating the Wnt5a transcription activator Pax2. Wnt5a treatment of irradiated FA mice enhanced HSC regeneration. Conversely, Wnt5a neutralization inhibited HSC regeneration after TBI. Wnt5a secreted by LepR+CXCL12+ BM stromal cells inhibited β-catenin accumulation, thereby repressing Prox1 transcription in irradiated HSCs. The detrimental effect of deregulated Wnt5a/Prox1 signaling on HSC regeneration was also observed in patients with FA and aged mice. Irradiation induced upregulation of Prox1 in the HSCs of aged mice, and deletion of Prox1 in aged HSCs improved HSC regeneration. Treatment of aged mice with Wnt5a enhanced hematopoietic repopulation. Collectively, these findings identified the paracrine Wnt5a/Prox1 signaling axis as a regulator of HSC regeneration under conditions of injury and aging.
Qiqi Lin, Limei Wu, Srinivas Chatla, Fabliha A. Chowdhury, Neha Atale, Jonathan Joseph, Wei Du