Hematopoietic Stem/progenitor Cells Research Articles

Targeting Cell Adhesion Molecules in IDH Mutant Myeloid Malignances

Neomorphic mutations in the genes encoding isocitrate dehydrogenase (IDH) are common in human cancers including myeloid malignancies. The oncometabolite (R)-2-hydroxyglutarate (R-2-HG) produced by mutant IDH promotes leukemia development by competitively inhibiting α-ketoglutarate (α-KG)-dependent dioxygenases, causing epigenetic dysregulation of histone and DNA methylation in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). FDA-approved allosteric inhibitors (ivosidenib and enasidenib targeting mutant IDH1 and IDH2 respectively) are used for treating newly diagnosed and/or relapsed/refractory AML patients with IDH mutations. However, the low response rate and primary resistance to IDHmut inhibitors are major clinical challenges. Moreover, acquired resistance to IDH inhibition can occur through multiple mechanisms, including R-2-HG-restoring second-site IDH mutations and the selection of sub-clonal mutations in RUNX1/CEBPA or RAS-RTK pathway. These findings underscore the importance of developing new and more effective approaches for the treatment of IDH-mutant MDS and AML patients. To identify mutant IDH-mediated alterations and actionable dependencies, we generated isogenic TF-1 myeloid leukemia cell lines containing endogenous, hotspot IDH mutations (IDH1R132H, IDH2R140Q, IDH2R140W, and IDH2R172K) by CRISPR/Cas9-mediated base-editing. By comparative transcriptomic (RNA-seq) and epigenomic (ATAC-seq and ChIP-seq) profiling of base-edited versus parental isogenic leukemia cells, we identified cell adhesion molecules, especially CD44, as the top upregulated genes in IDH-mutant leukemia cells. CD44 expression is significantly upregulated in IDH-mutant AML patient samples relative to normal CD34+ hematopoietic stem/progenitor cells (HSPCs) or other AML samples without IDH mutations. Increased CD44 expression is also associated with overall poorer survival and therapy resistance in independent AML cohorts. Importantly, we found that ectopic CD44 expression promotes cytokine-independent cell growth of TF-1 myeloid leukemia cells, resembling the effect observed upon expression of mutant IDH. Moreover, CD44 depletion led to decreased R-2-HG levels in base-edited IDH-mutant TF-1 leukemia cells, and impaired the propagation of IDH-mutant leukemia cells in vitro and in xenotransplanted recipient mice. These studies establish direct evidence that IDH mutations cause aberrant activation of cell adhesion molecules such as CD44, and CD44 is required for the propagation of IDH-mutant myeloid leukemia cells. Mechanistically, we found that the aberrantly activated CD44 is first cleaved by matrix metalloproteinases MMP2 and MMP9 for proteolytic release of its ectodomain from membrane, followed by γ-secretase-mediated intramembranous cleavage to produce the CD44 intracellular domain (CD44ICD). CD44ICD subsequently activates the pentose phosphate pathway (PPP) for the production of NADPH, a critical cofactor for mutant IDH enzymes to convert α-KG to R-2-HG. Hence, mutant IDH-mediated activation of CD44 functions to rewire intracellular metabolism to facilitate IDHmut-catalyzed R-2-HG production, supporting a model for mutant IDH-induced feed-forward oncogenic programs. CD44 also activates AKT/ERK signaling pathway to promote the proliferation and cytokine-independent growth of IDH-mutant leukemia cells. More importantly, primary and resistant IDH-mutant leukemia cells are sensitive to combination regimens consisting of IDHmut inhibitors and humanized monoclonal antibody specific for CD44 (RG7536) or MMP2/9 inhibitors (SB-3CT) invitro and invivo. Taken together, our findings uncover the cell adhesion molecule-mediated metabolic signaling as a new targetable dependency of IDH-mutant leukemia cells, with therapeutic implications for more effective treatment of primary and resistant IDH-mutant myeloid malignancies.

Jak2V617F Reversible Activation Shows an Essential Requirement for Jak2V617F in Myeloproliferative Neoplasms (MPNs)

JAK inhibitors (JAKi) improve symptoms and outcomes in MPNs; however, mutant allele burden does not substantively change with chronic therapy. Alternative MAPK signaling and/or cooperating mutations are thought to contribute to MPN cell persistence; however, we hypothesized current JAKi fail to effectively abrogate mutant JAK2 signaling. To that end, we developed a conditionally inducible mouse model allowing for sequential activation, then deletion, of Jak2VF from its endogenous locus using a Dre-rox/Cre-lox (Jak2RL) dual recombinase system (Figure 1A). Dre mRNA electroporation of lineage-negative UbcCreER-Jak2RL bone marrow (BM) was used to induce Jak2VFex vivo, followed by transplant into lethally-irradiated recipient mice in competition with Cd45.1 BM. Transplanted mice developed a highly penetrant MPN characterized by leukocytosis, polycythemia, hepatosplenomegaly, and BM megakaryocytic hyperplasia. Tamoxifen administration 12 weeks post-transplant to delete Jak2VF resulted in abrupt normalization of blood cell parameters, spleen weights, and serum pro-inflammatory cytokines, with a marked prolongation in overall survival (OS) compared to MPN control mice. Reduced peripheral blood (PB) and BM Cd45.2 mutant fraction was also observed, including within hematopoietic stem/progenitor cell (HSPC) compartments and to a greater degree than ruxolitinib (RUX) therapy. Notably, treatment with CHZ868, a type II JAKi, showed improvements in regard to hematocrit and spleen weight reduction on par with deletion, as well as significant reductions in BM HSPC mutant fractions, albeit to a lesser degree than with Jak2VF reversion (Figure 1B). Transplant of Jak2VF deleted BM failed to form phenotypic disease in secondary recipient mice consistent with loss of disease-propagating MPN stem cells. RNA-Seq on sorted LSKs following Jak2VF deletion showed loss of STAT5, MAPK, and MTORC1 target gene expression, as well as negative enrichment in TGFb and TNFa/NFkB pathways consistent with reduced pro-inflammatory and proliferative output. The alterations in MAPK signaling were supported by Ybx1 RT-PCR of BM cKit+ cells and BM pERK IHC, both of which were increased with RUX but potently suppressed with deletion suggesting improved JAK2 targeting can overcome MAPK-mediated persistence. The initial proximity of Jak2RLlox sites grants Cre-mediated recombination prior to Dre-mediated Jak2VF knock-in allowing for assessment of Jak2VF oncogenic dependency alone or in the context of co-occurring mutation. Given the known effects of Tet2 loss on enhanced MPN HSC fitness, we evaluated Tet2 loss on sustaining MPN with Jak2VF reversion. UbcCreER-Jak2RL/Tet2f/f mice were pulsed with TAM to knock-out Tet2 followed by sacrifice and Dre Jak2RL knock-in 6 weeks later. Mice transplanted with UbcCreER-Jak2VF/Tet2-/- cells exhibited enhanced leukocytosis, spleen weights and PB/BM chimerism compared to Jak2VF mice; however, Jak2VF reversion resulted in similar reductions in MPN disease parameters, including Cd45.2 fraction, suggesting Tet2loss does not dramatically alter dependence on mutant JAK/STAT signaling. Notably, while Jak2VF/Tet2-/- mutant cells displayed increased serial replating capacity, this was abrogated with Jak2VF reversion suggesting despite the enhanced fitness of Tet2-/- HSCs, Jak2VF creates a unique dependency that renders double-mutant cells susceptible to eradication with Jak2VF reversion. In sum, Jak2VF deletion abrogates MPN features, extends OS, and reduces mutant cell fraction with depletion of disease-sustaining MPN HSCs, either alone or in the setting of cooperating Tet2 loss. These data suggest JAK2VF mutant-selective inhibition offers greater therapeutic potential than current JAKi and a potential curative option for the treatment of MPN. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Analysis of CD45 Isoforms Displayed By Human Peripheral Blood Mononuclear Cells

CD45 is the most prevalent glycoprotein on the surface of mature leukocytes and hematopoietic stem/progenitor cells (HSPCs). Accordingly, this glycoprotein has been intensively interrogated since its discovery, and antibody-drug-conjugates (ADCs) and radioimmunoconjugates targeting the extracellular domain of CD45 have been explored in pre-clinical and clinical trials as a potential replacement of, or supplement to, existing myeloablative regimens for hematopoietic stem cell transplant (HSCT) preparation. Importantly, human CD45 consists of five distinct isoforms, resulting from assortment of three alternatively spliced exons (known as "A", "B", and "C"). Though monoclonal antibodies (mAbs) directed to each of these exons, and to the CD45 isoform bearing no such exons (i.e., the "CD45RO" isoform) are available, our understanding of the display of these distinct exons is grossly incomplete. To date, the most well-characterized leukocyte subset differentially expressing CD45 isoforms is the human T cell. By contrast, the human myeloid compartment's expression of specific CD45 isoforms has been largely unstudied. Given this, we sought to comprehensively interrogate CD45 isoform expression on human peripheral blood mononuclear cells following defined in vitro manipulations of the cells. CD4+ and CD8+ human T cells were isolated from peripheral blood of healthy donors and activated with anti-CD3 and anti-CD28 antibodies for 5 days. Before and after activation, we performed flow cytometry combined with western blot analysis to assess shifts to CD45RA, CD45RB, CD45RC, and CD45RO expression. We isolated baseline human CD14+ monocytes and utilized the same techniques to ascribe CD45 isoforms to resting human monocytes. Upon activation of human T cells, our analyses recapitulated the expected transition of these cells from CD45RA+ to CD45RO+. However, strikingly, all native and in vitro-activated human T cells universally react with anti-CD45RB mAb as determined by both flow cytometry and western blotting. Similarly, 100% of human CD14+ monocytes display CD45RB and CD45RO, as determined by flow cytometry and western blotting. The universal reactivity of human T cells with anti-CD45RB illustrates that canonical CD45RA-/CD45RO+ memory and activated T cells co-express the CD45RB isoform. In a similar manner, baseline human CD14+ monocytes co-express CD45RO and CD45RB isoforms. The capability to unveil the ubiquity of CD45RB isoform expression was enabled by concurrent use of exon-specific mAbs. These data indicate a previously unappreciated complexity in human CD45 isoform display, draw caution on simply stratifying CD45 expression on the basis of CD45RA or CD45RO phenotypes, and highlight a broader significance of the CD45RB isoform among human T cells and monocytes, and within the greater human hematologic system.

The Protein Interactome of DNMT3A, and Altered Interactions Caused By Missense Mutations Associated with AML and/or the DNMT3A Overgrowth Syndrome

DNA methylation mediated by the de novo DNA methyltransferases (DNMT3A and DNMT3B) is essential for normal embryonic development. Somatic loss-of-function or mutations in DNMT3A are common in individuals with clonal hematopoiesis, and are found in nearly 30% of normal karyotype AML patients; mutations at amino acid R882 represent a hotspot in AML, accounting for ~60% of DNMT3A mutations in AML. Acquired germline DNMT3A mutations are associated with the DNMT3A Overgrowth Syndrome (aka Tatton-Brown-Rahman Syndrome/TBRS), a leukemia susceptibility syndrome. Mutations at R882 have a dominant negative effect on WT DNMT3A, and cause a distinctive, focal, canonical DNA hypomethylation phenotype. Many non-R882 DNMT3A missense mutations also have a canonical hypomethylation phenotype in hematopoietic cells that is similar to that of haploinsufficiency caused by gene deletion (Smith et al, Nat. Comms, 2021), suggesting that they are also loss-of-function mutations. Although some non-R882 missensemutations may cause DNMT3A protein instability to cause loss-of-function (Huang et al, Can Disc 2022), others do not yet have a mechanism described. For this study, we investigated the hypothesis that missense mutations in DNMT3A may inactivate its methyltransferase activity by altering its ability to interact with proteins that influence its function. To better understand mechanisms involving altered protein interactions, we used a proximity labeling system using a highly active biotin ligase (TurboID/"TID"), fused in-frame to full length human DNMT3A1 at either the N-or C-terminus in an MSCV-based vector tagged with GFP. Vectors with either WT or mutant DNMT3A cDNAs were transduced into murine bone marrow progenitors, which were allowed to grow in culture for 2 days, enriched for GFP+ cells, and then labelled with biotin for 4 hours. Proteins within 10 angstroms of the TurboID-DNMT3A fusions were tagged with biotin, enriched with a streptavidin bead pulldown, and identified using mass spectrometry. We evaluated protein interactions with TurboID alone (n=9 independent experiments), wildtype DNMT3A (n=9), or with 21 different, recurrent missense mutations (7 TBRS mutations, 4 AML mutations, and 10 mutations found in both AML and TBRS patients, n=2-4 per mutation, with 53 total, independent samples) in mouse hematopoietic stem/progenitor cells (HSPCs). ETV3, RUNX1, DNMT3B, MBD1, GATA1, KDM5A, RB1, and 428 other proteins interacted specifically with wild type DNMT3A, compared to TurboID alone. Although some of these proteins had previously been identified as DNMT3A interactors, the vast majority were previously unknown (see Fig. 1A for the 50 strongest interactors). Altered DNMT3A protein interactions caused by missense mutations are shown for 10 selected proteins in Fig. 1B. Three mutations located in the PWWP domain of DNMT3A (G298W, I310N, and R326C) caused a loss of nearly all interactions, which was found to be due to mislocalization of DNMT3A to the cytoplasm by these variants (data not shown). Mutations known to cause DNMT3A instability (Huang et al, Can Disc 2022) including C583Y, R635W, Y660H, R736H, R749C, F902S and P904L, had increased interactions with ubiquitin ligases (Hectd3, Rnf31, Ube2v1, Smurf1, Trim8) which may target them for accelerated degradation (data not shown). The normal interaction between DNMT3A and DNMT3B was essentially eliminated by several mutations (I310N, R635W, Y660H, R736H) and decreased in several more (G298W, F902S and P904L) (Fig.1B). In AML cells, the dominant isoform of DNMT3B is DNMT3B3, which is missing exons that are essential for its methyltransferase activity. However, DNMT3B3 is known to interact with DNMT3A and, in doing so, increase its activity by acting as a chaperone. Therefore, the disruption of DNMT3B3 binding to DNMT3A may represent a novel mechanism for missense mutations to reduce DNMT3A activity. Finally, we used the same system to define DNMT3A protein interactions in human CD34+ cord blood cells (with DNMT3A WT, R736H, and R882H, and others in progress), which revealed high concordance with the mouse data. DNMT3A missense mutations can alter many protein interactions with DNMT3A, which can clearly reduce its methyltransferase activity by a number of mechanisms. These findings may suggest new approaches for restoring DNMT3A activity in patients with myeloid malignances. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comprehensive Profiling of Clinical JAK2 Inhibitors in Myeloproliferative Neoplasms

Since the approval of ruxolitinib (JAK1/JAK2) in 2011, two additional inhibitors, fedratinib (JAK2/FLT3) and pacritinib (JAK2/FLT3/IRAK1/CSF1R), have received FDA approval for myelofibrosis, and a new drug application for a fourth inhibitor, momelotinib (JAK1/JAK2/ACVR1/TBK1) has been submitted in June 2022. Although all four inhibitors potently suppress JAK2, the targeting of additional auxiliary targets may contribute to distinct responses observed in the clinic. Here, we take a comprehensive approach to assess the unique proliferative, transcriptional, and signaling pathways altered by these four JAK2 inhibitors in myeloproliferative neoplasms (MPN). In three human JAK2 mutant leukemic cell lines (HEL, UKE-1, SET2) and murine Ba/F3 EpoR-JAK2VF cells, ruxolitinib consistently showed the lowest IC50. Following, CD34+ hematopoietic stem/progenitor cells (HSPCs) from 8 MPN patients and 3 healthy donors were seeded for colony formation assays. Erythroid colonies were highly sensitive to ruxolitinib, fedratinib, and pacritinib inhibition. In contrast, momelotinib demonstrated greater relative sparing of erythroid colonies, consistent with clinical anemia amelioration, albeit potentially via a ACVR1/ALK2 and hepcidin-independent manner. Overall, greatest potency was demonstrated by pacritinib ex vivo, followed by ruxolitinib, fedratinib, and momelotinib. These findings were somewhat distinct from in vitro assays, likely reflecting additional underlying biology of human HSPCs. We then performed high dimensional mass cytometry to assess signaling changes at single cell resolution across different cell populations in 9 MPN patient samples and 3 healthy donors treated ex vivo with JAK inhibitors. We observed potent suppression of pSTAT1, pSTAT3, and pSTAT5 in CD34+ cells, with the 4 JAK2 inhibitors exerting similar degrees of inhibition. In B cells, pacritinib suppressed IκBα, pERK, and pCREB levels, while momelotinib inhibited pTBK1. Similar observations were noted in T cells, which in addition exhibited suppression of pSTAT1 and pSTAT3. These findings indicate distinct suppression responses across specific cell types and further highlight nuances beyond sole suppression of JAK-STAT signaling by these inhibitors. RNA-sequencing of HEL cells treated with the 4 JAK2 inhibitors also identified unique alteration of expression profiles and pathways (Fig. A). Differential gene expression (DEG) analysis revealed pacritinib inducing the greatest transcriptional alterations after 4 hours (3,121 genes) and ruxolitinib (1,969 genes) following 48 hours treatment duration. Only 82 and 103 DEGs relative to control were shared across the 4 JAK2 inhibitor treatment at 4 and 48 hours, respectively. Additional pathway analysis revealed at least 3 out of 4 inhibitors demonstrating capacity to suppress 1) JAK-STAT signaling, 2) inflammatory response, and 3) TNF signaling via NFκB pathways. Upregulation of multiple metabolic pathways was observed such as oxidative phosphorylation by momelotinib and pacritinib, potentially reflecting adaptive transcriptional feedback, as Seahorse Mito Stress assays revealed strongest suppression of oxygen consumption rate of PBMCs mediated by pacritinib and fedratinib. Lastly, we compared the in vivo potency of JAK2 inhibitors using patient-derived xenograft (PDX) models (Fig. B). Engrafted immunodeficient NSGS mice were treated with JAK2 inhibitors for 4 weeks across 2 sets of PDX experiments with CD34+ cells from unique JAK2 and CALR mutant donors. Pacritinib demonstrated greatest potency in reduction of peripheral blood and bone marrow human CD45+ leukemic cells, followed by ruxolitinib, and fedratinib/momelotinib. Vehicle treated mice were moribund by week 3 of treatment and survival was significantly prolonged with all JAK2 inhibitor treatments, with all animals surviving in the pacritinib treatment group after 4 weeks of treatment at end point. Despite all four JAK2 inhibitors demonstrating affinity in suppressing JAK-STAT signaling, our assays reveal varying degrees of alteration of transcriptional, proteomic, and metabolic profiles. In conjunction with comparative assessment of in vivo activity in PDX models, these findings provide insights with potential relevance to distinct responses observed with these inhibitors in the clinic and may help guide the use of specific JAK2 inhibitors in combination studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Recovery of Highly Functional Human Hematopoietic Stem and Progenitor Cells from 27-Year Old Cryopreserved Umbilical Cord Blood

Umbilical cord blood (CB) is a promising source of hematopoietic stem cells (HSCs) and progenitor cells (HPCs) for hematopoietic cell transplantation (HCT). CB is cryopreserved following collection, allowing for the amassment of nearly 800,000 CB units in public banks and an estimated 4 million in private banks. Cryopreservation of CB is essential for successful CB transplantation, and it is critical to understand how long CB can be stored with efficient recovery of engraftable HSCs. Prior work demonstrated efficient recovery of engraftable HSCs from 10-, 15-, and 21-year old cryopreserved CB. Here, we demonstrate efficient recovery of highly engraftable HSCs from 27-year old cryopreserved CB units. 3 CB units cryopreserved for >27 years were thawed with clinical protocols, and total nucleated cells were recovered with 76-87% viability. 3-6 fresh CB units were used as a comparison for cryopreserved units. Immunophenotyping assays to determine numbers of HSCs/HPCs showed that cryopreserved CB units had on average 4.25x103 CD34+ cells per 1x106 nucleated low density CB cells (LDCB), similar to fresh CB with 4.55x103CD34+ per 1x106 LDCB. Within the CD34+ population, cryopreserved CB when compared to fresh CB had on average equal or greater numbers of stringently immunophenotypically defined HSCs (9.1x103 vs 6.2x103), multipotent progenitors (MPPs) (2.1x105 vs 8.6x104), and multilymphoid progenitors (MLPs) (1.9x104 vs 1.0x104) per 1x106 CD34+ cells. Conversely, there were fewer numbers of common myeloid progenitors (CMPs) (2.9x104 vs 1.1x105), megakaryocyte-erythroid progenitors (MEPs) (4.5x104 vs 1.0x105), and granulocyte macrophage progenitors (GMPs) (4.8x104 vs 1.4x105). Colony forming unit (CFU) assays demonstrated that cryopreserved CB units exhibited lower functional HPC numbers per 1x106 CD34+ enriched cells including CFU-GM (1.9x104 vs 8.3x104), BFU-E (1.2x104 vs 3.4x104), and CFU-GEMM (1.1x104 vs 5.8x104). In ex vivo analysis of HSC/HPC proliferative capacity, cryopreserved CB units showed similar or greater capacity for expansion in growth stimulating media compared to fresh CB, including HSCs (7.7-fold vs 6.2-fold increase), MPPs (3.5-fold vs 4-fold increase), MLPs (44-fold vs 1.7-fold increase), CFU-GM (29-fold vs 13.3-fold increase), and CFU-GEMM (28.6-fold vs 4-fold increase). For in vivo functional HSC analysis, 10,000 CD34+ enriched cells were transplanted to sublethally irradiated NSG mice. Cryopreserved CB units exhibited early and late human engraftment in peripheral blood (PB) at months 1, 2, 4, and 5. Bone marrow (BM) human chimerism at month 5 ranged from low (0.5%) to robust levels of engraftment (52.7%). Limiting dilution analysis revealed that 2 cryopreserved CB units had similar SCID repopulating cell (SRC) frequency, ranging from 314 to 1392 SRCs per 1x106 CD34+ cells while the third had a much lower 65 to 393 SRCs per 1x106 CD34+ cells. The third CB unit also exhibited lower average human chimerism in the BM (0.55%) than the other 2 units (15% and 16.2%). However, the SRC frequencies in all 3 CB units are similar to those in fresh CB units published by our group and others (range 51 to 929 SRCs per 1x106 CD34+ cells). Secondary transplants showed that all 3 CB units have secondary repopulation capacity demonstrated by human chimerism in PB. These data suggest functional HSCs with short- and long-term engrafting and sustained self-renewal capacity can be cryopreserved for at least 27 years. We show that different cryopreserved CB units contain variable functional HSC frequency. Similar results in ex vivo characterization suggest cryopreservation is not the cause of these functional differences, but rather there were functional differences in the CB units prior to cryopreservation. This underscores the need to have reliable measures of CB functionality prior to cryopreservation for more efficient selection of donor units and to test potential effects of cryopreservation on individual units. Transcriptomic studies are ongoing to determine molecular programs that may contribute to differences in SRC frequency. These data will provide insight into mechanistic explanations for differences in HSC function in different CB units. Authors’ note: This project was conceived by the authors prior to the death of Hal E. Broxmeyer. Dr. Broxmeyer initiated the study as a continuation of his life's work demonstrating the utility of CB for HCT. This work is dedicated to Hal.

Sitagliptin Alleviates Excessive Autophagy in Macrophages in Immune Thrombocytopenia Patients to Promote Megakaryopoiesis Via the AMPK/mTOR Pathway

Introduction: The pathogenesis of ITP is complex and mainly manifests as autoantibody-mediated platelet destruction and impaired platelet production. Autophagy is a highly conserved cellular process that eliminates damaged organelles and promotes the degradation of misfolded proteins. Appropriate autophagy exerts a protective effect, while excessive autophagy may lead to cell death and increased inflammation. The specific role of bone marrow macrophages (BM MΦ) in regulating megakaryopoiesis in ITP patients has not been elucidated. Sitagliptin, a DPP-4 inhibitor, was reported to play a key role in the production and engraftment of hematopoietic cells by modulating SDF-1α and several colony-stimulating factors, thus enhancing hematopoietic stem and hematopoietic progenitor cell recovery. However, whether sitagliptin has the potential to promote megakaryocyte maturation and platelet production in ITP is unclear. Therefore, this article explores the effect of macrophage autophagy on megakaryopoiesis in ITP patients and investigates its underlying role in the pathogenesis of ITP in order to explore whether sitagliptin could be a potential treatment for ITP. Methods: BM MΦ and CD34+ cells were isolated from ITP patients and healthy control subjects. Excessive autophagy in bone marrow macrophages from ITP patients was confirmed by Western blotting and transmission electron microscopy. PCR and Western blotting were used to assess the expression of A20 and autophagy-related AMPK/mTOR/ULK1 pathway components in ITP-MΦ. Vectors carrying A20 cDNA/TNFAIP3-specific shRNA were transduced into the BM MΦ from either healthy control subjects or ITP patients. The different BM MΦ populations were separately cocultured with CD34+ cells. We compared the ability of BM MΦ to support megakaryocytopoiesis by determining the relative percentages of MKs at different stages using flow cytometry as well as CFU counts of megakaryocytes and platelets in culture. The mechanism by which sitagliptin normalized the impaired function of ITP-MΦ was evaluated in vitro. A prospective single-arm clinical cohort study was designed to explore the efficacy and safety of sitagliptin in the treatment of corticosteroid-resistant or relapsed ITP. Results: The expression of LC3-II/LC3-I and Beclin1 increased in ITP-MΦ, and the expression of P62 decreased. The number of autophagic vesicles also increased, indicating enhanced autophagy in ITP-MΦ. Transcriptome sequencing and untargeted metabolomics revealed that macrophage autophagy-related pathways were involved in the pathogenesis of ITP. Further studies confirmed that ITP-MΦ regulated megakaryopoiesis, which was dependent on the A20-mediated autophagy-related AMPK/mTOR/ULK1 pathway. Inhibition of A20 expression with A20-shRNA inhibited the AMPK/mTOR/ULK1 pathway and alleviated excessive autophagy in ITP-MΦ, thus increasing the number of megakaryocytes, the proportion of hyperploid megakaryocytes and the number of platelets produced in the ITP-MΦ coculture system. Inhibition of autophagy with the autophagy inhibitor 3-MA reversed the A20 overexpression-induced excessive autophagy in MΦ and normalized the differentiation and maturation of megakaryocytes. Inhibiting the AMPK/mTOR/ULK1 signaling pathway with the AMPK inhibitor Compound C reversed the A20 overexpression-induced increase in the expression of AMPK/mTOR/ULK1 autophagy pathway components in MΦ and normalized megakaryopoiesis. Sitagliptin was found to inhibit the expression of A20 and AMPK/mTOR/ULK1 autophagy pathway components in ITP-MΦ in vitro. Sitagliptin improved the ability of ITP-MΦ to promote the differentiation and development of megakaryocytes and platelet production. Sitagliptin was administered to a total of 56 patients with corticosteroid-resistant or relapsed ITP. Complete response and overall response were observed in 11 (19.6%) and 21 patients (37.5%), respectively. Conclusions: A20 mediated the AMPK/mTOR/ULK1 signaling pathway to regulate autophagy in ITP-MΦ and affected megakaryopoiesis and platelet formation. Sitagliptin could restore the proper function of the A20-mediated AMPK/mTOR/ULK1 signaling pathway in ITP-MΦ, thus promoting megakaryopoiesis. Sitagliptin resulted in a sustained response in patients with corticosteroid-resistant/recurrent ITP and exhibited good safety. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Arcch: Improving the Performance of Clonal Hematopoiesis Variant Calling and Interpretation Using a Consensus Based Approach

Introduction: The acquisition of somatic mutations in hematopoietic stem and progenitor stem cells with resultant clonal expansion is known as clonal hematopoiesis (CH). CH is linked to a higher risk of hematologic malignancies and other adverse health outcomes. The prevalence of CH is dependent on sequencing technique. With ultra-high-depth sequencing, CH can be detected in most adults. However, CH variant calling at very low variant allele fractions (VAF) is challenging due to difficulty in distinguishing low frequency CH mutations from sequencing artifacts. Here we develop and validate a novel CH variant calling approach that combines ensemble based variant calling with advanced artifact filtering and outperforms commonly used somatic variant callers. Methods: We sequenced a tumor-normal dilution series from six AML patients and 27 technical controls with a targeted panel utilizing unique molecular indexes that included nine common CH genes (DNMT3A, TET2, ASXL1, TP53, PPM1D, JAK2, SF3B1, SRSF2) with a mean unique coverage of 18,268x. In total we identified 32 mutations in these six samples validated by an independent sequencing panel performed for the purpose of clinical testing. The variant allele fraction (VAF) in the resulting tumor-dilution samples ranged from 0.1%-50% VAF. Results: For higher VAF mutations (>5%), Mutect2, Lofreq, and Vardict showed reasonable sensitivity (range: 0.70-1.00) and positive predictive values (PPV) (range: 0.80-1.00). However, at lower VAF, PPV was poor; for mutations between 0.1-0.4% VAF, the PPV ranged between 0.01-0.22. We applied additional filters based on sequencing quality, depth, regional complexity, and an empiric estimate of sequencing error at the position of a given called variant using technical controls. Taking the consensus of three callers and including our additional filters, we not only retained sensitivity and PPV for higher VAF variants above 1% (0.92 sensitivity, 0.95 PPV) but improved the PPV with retained sensitivity for variants between 1-0.1% VAF (0.91 sensitivity, 0.83 PPV). Applying our consensus approach to blood samples drawn from 31 healthy individuals sequenced using the same targeted panel with variants validated using an orthogonal approach, we saw excellent sensitivity and PPV across a wide range of VAFs (Sensitivity 0.99 [>1% VAF], 0.92 [0.2-1% VAF], and 0.82 [0.1-0.2% VAF]; PPV 1.00 [>1% VAF], 0.98 [0.2-1% VAF], 0.97 [0.2-0.1% VAF]). We compared our consensus calling and advanced filtering approach to a machine learning model. A decision-tree-based ensemble machine learning algorithm, XGBoost, was trained on the tumor-dilution samples with validation using 31 blood samples with orthogonal sequencing. The variant caller output (allele counts, flags, etc.), additional false positive filtering metrics, and statistical results from the technical controls were used as features. Sensitivity and PPV was high for mutations above 1% VAF (0.97 sensitivity and 0.82 PPV) with the performance declining for lower VAF mutations (0.72 sensitivity and 0.65 PPV). Thus, a machine learning approach did not outperform consensus calling with advanced artifact filtering. Finally, we developed a custom annotation pipeline for CH variant pathogenicity classification incorporating previously reported CH mutations in large cohorts, cancer somatic mutation frequency databases and curated cancer driver knowledge databases. Conclusion: We developed ArCCH, an advanced artifact filtering and consensus CH calling pipeline that includes custom annotation features to facilitate CH pathogenicity determination. Our results show that our consensus approach with advanced error correction filters substantially improves the performance of CH variant calling beyond commonly used single variant callers. This end-to-end flexible workflow package is publicly available on Terra and will facilitate future CH studies.

Expression of Jak2 R1063H Leads to Altered Epor/Tpor Signaling, Changes in HSC Fitness and Represents Increased Risk of Thrombosis

Non-canonical weakly activating germline JAK2 variants may predispose progenitor cells to acquire JAK2 V617F during the evolution of myeloproliferative neoplasms (MPN) (Lanikova et al. Blood 2016;128(18):2266) and represent risk factors for MPN transformation into AML (Benton et al. Cancer 2019;125:1855). We have previously described and functionally characterized the JAK2 R1063H germline variant, cooperating with the JAK2 E846D variant in a case of hereditary MPN with erythrocytosis and megakaryocytic atypia (Kapralova et al. Blood 2016;128:1418) or with the acquired JAK2 V617F causing increased JAK2 signaling and higher disease severity in patients with essential thrombocythemia (Mambet et al. Blood 2018;132:2695). The mouse model bearing Jak2 R1063H mutation was created using microinjection of synthesized ssODNs containing the R1063H mutation, sgRNA mRNA and Cas9 protein into C57BL6/N-derived zygotes. In order to intensify a possible effect of the mutation, we bred and analyzed the knock-in Jak2 R1063H homozygous mice. The mice are born in normal (expected) frequency and display increased platelets (PLT) counts when compared to wild-type (wt) mice during life-span. Despite increased PLT counts, the plasma thrombopoietin level is normal whereas erythropoietin levels are increased without marked erythrocytosis. The preliminary co-immunoprecipitation experiments with human erythropoietin and thrombopoietin receptors (EPOR/TPOR) and JAK2 variants suggest increased TPOR and decreased EPOR binding with JAK2 R1063H kinase, possibly explaining the hematological phenotype. Also experiments with humanized mouse model of EPOR (Divoky et al. Proc Natl Acad Sci USA 2001;98:986) crossed with Jak2 R1063H mice supported these findings, where anemia due to hypoactivation of wt human EPOR signaling is more severe in Jak2 R1063H mice than in a control group and mild thrombocytosis is still present in the double mutant mice. The Jak2 R1063H model displays increased number of long-term hematopoietic stem cells (HSC) suggesting increased cycling in this population at young age (3 months) and shift to increased short-term HSC and multipotent progenitors with aging (12 months). The immunophenotypically-defined myeloid progenitor cells (LK, Lin-/Sca1-/cKithigh) are statistically increased in Jak2 R1063H mice due to disproportionately increased megakaryocytic/erythroid progenitors (MEP) over other myeloid progenitors at 3 months of age. The LK compartment is compromised in old Jak2 R1063H mice due to substantial myeloid bias by disproportionately increased CMP cells and marked decreased MEP cells, explaining the more pronounced anemia of aging in these mice. Some of these features are reminiscent of aging-associated myeloid and megakaryocytic bias, increased LKs, and relative exhaustion of progenitors with an erythroid lineage-priming program in aged bone marrow (BM) of a mouse model of primary familial and congenital polycythemia (PFCP) due to gain-of-function EPOR (Kralova et al, Am J Hematol 2022; doi: 10.1002/ajh.26658). However, the gain-of-function EPOR-introduced changes into the BM microenvironment are not associated with augmented hypoxia signaling and systemic inflammation and do not represent permanent challenges that would contribute to an increased incidence of myeloid malignancies in PFCP. In this regard, it will be interesting to compare these characteristics with the possible predisposing potential of Jak2 R1063H; further analysis of proliferative history of Jak2 R1063H HSC populations and expression profiling are ongoing. In the cohort of Jak2 R1063H mice, we also observed a relatively high frequency of sudden death in mice with median overall survival of 215 days (5/15) in comparison with the control group (0/15). We therefore measured the levels of D-dimers as a marker of thrombosis and found it statistically increased in the Jak2 R1063H group. Interestingly, in a WES study of patients with venous thromboembolism, the JAK2 R1063H was identified in one case, being considered a probable disease-causing variant (Lee at el. Blood Adv 2017;1:1224). In conclusion, our results confirm a direct effect of germline Jak2 R1063H mutation on EpoR/TpoR signaling but also on HSC biology, suggesting that the variant may represent a risk factor for BM remodeling in the process of myeloid cell transformation. Grant support: Czech Health Research Council NU21/03/00338.

Clinical Significance of IGH Clonality Analysis with RNA Sequencing in B-Cell Precursor Acute Lymphoblastic Leukemia

Background: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common pediatric malignancy. Using minimal residual disease (MRD) and genetic subtypes, risk stratification algorithms have considerably improved clinical outcomes. However, despite being in a better prognostic group, a non-negligible number of relapses occur, and prognostic markers for these patients are required for more effective treatment strategies. RNA sequencing (RNA-seq) is a useful tool for detecting patient-specific immunoglobulin heavy chain (IGH) junctional sequences (Li et al., Leukemia. 2020), which can be informative for evaluating the nature of leukemic clones. Here, we analyzed IGH clonality of pediatric BCP-ALL using RNA-seq data and revealed its validity for evaluating disease characteristics and impact on prognosis. Methods: We analyzed RNA-seq data of 136 pediatric BCP-ALL samples at diagnosis and 46 samples at relapse from the TARGET ALL Expansion Phase 2 cohort. Using MiXCR (Bolotin et al., Nat Methods 2015) and Vidjil (Duez et al., PLoS One 2016), we detected IGH complete VDJ and incomplete DJ rearrangements. Then, we estimated the expected number of IGH rearrangements corresponding to each read count (E-value) by power-law model. Finally, we identified leukemic "disease clones” with E-values less than 0.01 (Fig. 1a). Results: First, we detected a total of 55,518 IGH clones, of which 48,555 were complete rearrangements and 6,963 were incomplete. In total, 351 disease clones (median 2, range 0-8 per sample) were identified by power-law model: 193 clones were complete rearrangements, and 158 clones were incomplete. Interestingly, the numbers and compositions (complete or incomplete rearrangements) of disease clones varied significantly between samples. Thus, we defined three clonality patterns and classified each sample based on identified disease clones: "Undetectable disease clones (UDC)” if the sample had no disease clone (n=18); "Incomplete Disease Clones (IDC)” if there were one or more disease clones with incomplete rearrangements (n=72); and "Complete Disease Clones (CDC)” if all disease clones of that sample have complete rearrangements (n=46). Gene Set Enrichment Analysis (GSEA) indicated a strong enrichment for hematopoietic stem cell (HSC) and multipotent progenitor (MPP) signatures in UDC and pro-B cell signatures in IDC. Similarly, CIBERSORTx (Newman et al., Nat Biotechnol 2019) showed higher proportions of HSCs, MPPs, and pre-pro B cells in UDC and pro-B cells in IDC. Furthermore, there was a significant enrichment in pathways promoting cell cycle progression in IDC. Of note, in high hyperdiploid (HHD) cases, which are typically associated with a favorable prognosis, event-free survival (EFS) was significantly lower in UDC and IDC (p=0.01) (Fig. 1b). Moreover, even in MRD-negative HHD patients, those with UDC and IDC had significantly worse EFS (p=0.01) and overall survival (p=0.03) (Fig. 1b). Finally, we compared the primary and relapse clones in 46 patients. As a result, 37 patients had one or more disease clones at diagnosis, and 29 (78%) had these primary disease clones at relapse. On the other hand, 9 (24%) patients showed relapse-specific disease clones, which should lead to relapse in these cases. The other 9 primary patients with UDC had no disease clone also at relapse. Discussions: We identified three clonality patterns, UDC, IDC, and CDC based on RNA-seq analysis of the IGH rearrangement status. According to the findings of GSEA and CIBERSORTx, leukemic cells of UDC, IDC, and CDC patients have nature of progenitors to pre-proB cells, pro-B cells, and more mature cells, respectively. These findings are consistent with the knowledge that IGH rearrangement occurs during pro-B to pre-B maturation in normal B-cells. Patients with immature clonality status, UDC and IDC, were associated with poor prognosis in high hyperdiploid subtype and could be a potential prognostic marker in MRD-negative patients. In addition, we revealed that primary disease clones contributed to relapse in 78% of cases, while relapse-specific clones were implicated in relapse in 24% of cases. Additionally, UDC clonality pattern was carried over at relapse. These findings shed light on the mechanisms of leukemic clonal evolution and relapse, and suggest that the status of IGH rearrangement may serve as a novel prognostic marker in BCP-ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Long-Term Patient-Reported Outcomes Following Treatment with betibeglogene autotemcel in Patients with Transfusion-Dependent β-Thalassemia

Introduction: Patients (pts) with transfusion-dependent β-thalassemia (TDT) have increased morbidity and mortality and reduced quality of life (QoL) compared with the general population owing to the mental, physical, and time demands of chronic transfusions and iron chelation. Autologous gene therapy with betibeglogene autotemcel (beti-cel) therapy uses a modified β-globin gene that establishes stable production of functional adult hemoglobin (Hb), thus allowing for transfusion independence (TI). Pts with TDT were treated with beti-cel in 2 completed phase 1/2 studies (HGB-204, HGB-205) and 2 ongoing phase 3 studies as of Aug 2021 (HGB-207, HGB-212). After 2 years of follow-up, pts could enroll in a long-term follow-up study (LTF-303 [NCT02633943]) for up to an additional 13 years. QoL improvement in the 1-2 years post beti-cel treatment was previously described. Here, we evaluate long term (3 years post-treatment) patient reported outcomes after beti-cel treatment in pts with TDT enrolled in LTF-303. Methods: Pts with TDT underwent hematopoietic stem progenitor cell collection followed by myeloablative busulfan conditioning and beti-cel infusion. TI is defined as weighted average Hb ≥9 g/dL without packed red blood cell transfusions for ≥12 months. Generic health related QoL (HRQoL) over time was assessed using the Pediatric Quality of Life Inventory (PedsQL; range, 0-100) for pediatric and adolescent pts (<18 years old [YO]). Short Form-36 Health Survey Physical Component Summary and Mental Component Summary (SF-36 PCS and MCS, respectively; range 0-100), and the EuroQol (EQ-5D-3L) visual analog scale (VAS; range 0-100) composite index score (range 0-1) for adult (≥18 YO) pts. HRQoL was assessed at baseline (BL), Months (M) 6, 12, 18, 24 (in the parent HGB studies), and M 36, 48, and 60 (in LTF-303) after beti-cel infusion. Pts were excluded from HRQoL analyses if BL or M36 HRQoL assessment was missing as of data cut (Aug 2021). Higher scores indicated improvement in HRQoL. A bespoke questionnaire assessing β-thalassemia and pt-centric aspects of daily living (i.e., ability to work or attend school, physical activity, and perceived benefit from treatment) was completed at M 36. Results: As of August 18, 2021, 57 pts were enrolled in LTF-303 (phase 1/2: 22 pts; phase 3: 35 pts), of which 26 (45.6%) were pediatric / adolescent pts and 31 (54.4%) were adult pts. Among pts <18 YO with PedsQL data who achieved TI, the mean (standard error (SE)) PedsQL total score (population norm = 80.9) increased from 77.4 (3.4) at BL (n=19) to 92.1 (4.2) at M 36 (n=4). Among adult pts with SF-36 data who achieved TI, the mean (SE) SF-36 PCS and MCS scores (US general population norm = 50) increased from 53.8 (1.4) and 50.9 (1.7), respectively, at BL (n=12) to 55.7 (1.7) and 56.3 (1.4) at M 36 (n=9), respectively. The mean (SE) EQ-5D-3L Composite Index and VAS scores increased from 0.92 (0.04) and 85.2 (3.0), respectively, at BL (n=12), to 0.95 (0.03) and 91.4 (2.2) at M 36 (n=9) (Table 1). Data from the M 36 testimonial questionnaire (Figure 1) showed the ability to seek employment or be employed, increased from 68.8% (11/16) of pts at BL to 93.8% (15/16) at M 36. There was a reduction in school absences at M 36 (44.4% [4/9] missing school) compared with BL (83.3% [15/18] missing school). 50% (10/20) of pts still required management of disease symptoms at M 36. 80% (16/20) of pts reported improvement in physical activity at M 36. 100% (19/19) of pts reported an overall benefit from undergoing treatment with beti-cel. Conclusions: Published data shows beti-cel is potentially curative in pts with TDT, defined as having TI with normal or near-normal Hb. It was previously shown that with high-range QoL at BL, QoL improved at 1-2 years post-beti-cel. The updated data indicates durability of QoL improvement through year 3. A trend towards a positive impact on employment, school attendance, physical activity, and other aspects of life is also observed. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Novel Combination of Clinical Menin Inhibitor Ziftomenib and the Nuclear Export Inhibitor Selinexor Synergistically Inhibit MLL-r AML

PURPOSE: Menin is a scaffold protein that tethers histone-lysine-N-methyltransferase MLL1 (KMT2A) to chromatin by binding to the menin binding domain (MBD) of KMT2A. The N-terminus of the KMT2A gene recombines with a range of genes to form more than 80 fusion proteins (FP) in MLL-rearranged (MLL-r) AML. In myeloid stem progenitor cells, the menin-KMT2A complex modulates the expression of the leukemogenic homeobox A9 (HOXA9) gene, as well as its co-factor, MEIS1. Menin inhibitors evict menin from the chromatin and reduce KMT2A and KMT2A-FP binding to their targets in MLL-r leukemia, breaking the differentiation block and inducing tumor regression. Aberrant nuclear export is common in cancer, resulting in anomalous localization of various proteins, including tumor suppressor proteins (TSPs). Blocking XPO1/CRM1 using a selective inhibitor of nuclear export (SINE) compound selinexor has been shown to have robust anti-leukemia activity. In the present study, we hypothesized that inhibition of menin and nuclear export would synergistically suppress AML cell proliferation and survival. We have used the clinical-stage menin inhibitor ziftomenib and selinexor to simultaneously target menin-KMT2A protein-protein interactions and nuclear export. METHODS: To assess growth inhibition, an ATP-based cell proliferation assay was used. Combination synergy was determined using CalcuSyn Version 2.0 synergy software. Stem-like progenitor cells were isolated using the StemSpan CD34+ expansion kit (StemCell Tech). Colony formation efficiency was determined using a Methocult assay (StemCell Tech). Gene and protein expression and cell death were detected using quantitative real-time PCR, western blotting, and annexin-V/PI fluorescence-activated cell sorting, respectively. RESULTS: The clinical candidate ziftomenib, in combination with selinexor, synergistically inhibited the growth of MLL-r AML cell lines (MV4;11, MOLM13, and SEM) (CI<1) [Fig.1A]. The combination of menin and XPO1 inhibitors significantly suppressed colony formation of CD34+ MLL-r progenitor cells derived from primary patient samples but was not toxic to normal CD34+ human hematopoietic progenitor stem cells (HPSCs). Menin inhibitor treatment caused the downregulation of menin protein along with its downstream targets HOXA9, MEIS1, and FLT3 in western blot analysis. Interestingly, the nuclear export inhibitor selinexor also reduced menin, HOXA9, and MEIS1 protein levels. The combination of menin and XPO1 inhibitors enhanced menin down expression, suggesting a molecular basis for the synergy between the two compounds [Fig 1.B]. Ziftomenib suppressed the expression of menin in a time-dependent manner, which is evident as early as six hours of treatment. This combination significantly enhanced both early and late apoptotic cell death in MLL-rearranged AML cells compared to either compound alone. We have performed total RNA sequencing and observed differential expression of certain mRNAs and micro RNAs (miRs). The TMEM191B, SH3TC1, and SULT1C2 mRNAs were upregulated by KO539 and Selinexor, which are enhanced further by combination treatment. On the contrary, CENPK, CKS2, KIF18A, and H2BC4 mRNAs were downregulated by a single agent or combination. Among miRs, hsa-miR-34a-5p, hsa-miR-142-3p, hsa-miR-26b-5p were upregulated and hsa-miR-33b-3p, hsa-miR-4454, hsa-miR-210-3p and hsa-miR-130b-3p [reported to be an essential lineage-specific codriver of MLL-AF4 leukemia] were downregulated by single agent or combination. Such mRNAs and miRs panels could serve as biomarkers for treatment response. CONCLUSION: These preclinical findings demonstrate that simultaneous inhibition of the menin-KMT2A interaction and nuclear export could be a viable strategy for the treatment of MLL-r AML. Ongoing experiments with CDX and PDX using MLL-r models and functional analysis using total transcriptome sequencing and proteomics will be presented at the ASH meeting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

3D organoid modeling of extramedullary hematopoiesis.

Under certain clinical and experimental conditions hematopoiesis occurs in other site than bone marrow (BM), such as the liver. Here, we develop a 3D organoid that mimics several components of the hematopoietic niche present during liver extramedullary hematopoiesis. To evaluate the capacity of a 3D hematopoietic organoid (3D-HO) to function as a hematopoietic like-niche allowing for blood cell production outside of the BM. The 3D-HO is constituted by liver sinusoidal endothelial cells (LSEC) as the stromal component, BM isolated from 5-FU treated mice (FU-BMCs), collagen microspheres and plasma clot as scaffolds. The ability of the 3D-HO to support the survival and functionality of FU-BMCs was investigated by using confocal microscopy, histology analysis, flow cytometry, and clonogenic assays. After 15 and 30 days, post-ectopic implantation, histological studies of the 3D-HO showed the presence of cells with myeloid and lymphoid lineage morphology. Flow cytometry analysis of these cells showed the presence of cells expressing hematopoietic stem progenitor cells (HSPC) (Sca-1+/c-Kit+), myeloid (Gr-1+) and lymphoid (B220+ and CD19+) markers. Clonogenic assays showed that cells from the 3D-HO formed hematopoietic colonies. Expression of the Sry gene by cells from the 3D-HO, implanted for 30 days in female mice, indicated that male donor cells persist in this model of extramedullary hematopoiesis. The 3D-HO constitutes an extramedullary hematopoietic-like niche which supports the survival and functionality of FU-BMCs. It may constitute an efficient model for investigating, in vitro and in vivo, those factors that control hematopoiesis outside BM.

The PIP4K2 inhibitor THZ-P1-2 exhibits antileukemia activity by disruption of mitochondrial homeostasis and autophagy

The treatment of acute leukemia is challenging because of the genetic heterogeneity between and within patients. Leukemic stem cells (LSCs) are relatively drug-resistant and frequently relapse. Their plasticity and capacity to adapt to extracellular stress, in which mitochondrial metabolism and autophagy play important roles, further complicates treatment. Genetic models of phosphatidylinositol-5-phosphate 4-kinase type 2 protein (PIP4K2s) inhibition have demonstrated the relevance of these enzymes in mitochondrial homeostasis and autophagic flux. Here, we uncovered the cellular and molecular effects of THZ-P1-2, a pan-inhibitor of PIP4K2s, in acute leukemia cells. THZ-P1-2 reduced cell viability and induced DNA damage, apoptosis, loss of mitochondrial membrane potential, and the accumulation of acidic vesicular organelles. Protein expression analysis revealed that THZ-P1-2 impaired autophagic flux. In addition, THZ-P1-2 induced cell differentiation and showed synergistic effects with venetoclax. In primary leukemia cells, LC-MS/MS-based proteome analysis revealed that sensitivity to THZ-P1-2 is associated with mitochondrial metabolism, cell cycle, cell-of-origin (hematopoietic stem cell and myeloid progenitor), and the TP53 pathway. The minimal effects of THZ-P1-2 observed in healthy CD34+ cells suggest a favorable therapeutic window. Our study provides insights into the pharmacological inhibition of PIP4K2s targeting mitochondrial homeostasis and autophagy, shedding light on a new class of drugs for acute leukemia.

Uncovering the emergence of HSCs in the human fetal bone marrow by single-cell RNA-seq analysis.

During fetal development, human hematopoietic stem cells (HSCs) colonize the bone marrow (BM), where they self-renew and sustain hematopoiesis throughout life; however, the precise timepoint at which HSCs seed the BM is unclear. We used single-cell RNA-sequencing to map the transcriptomic landscape of human fetal BM and spleen hematopoietic stem/progenitor cells (HSPCs) and their microenvironment from 10 to 14 post-conception weeks (PCWs). We further demonstrated that functional HSCs capable of reconstituting long-term multi-lineage hematopoiesis in adult NOG mice do not emerge in the BM until 12 PCWs. In contrast, functional HSCs were not detected in the spleen by 14 PCWs. By comparing the niche-HSPC interactions between BM and spleen, we identified ligand-receptor pairs likely to be involved in fetal HSC migration and maintenance. Our work paves the way for research into the mechanisms underlying HSC colonization in human fetal BM and provides invaluable resources for future studies on HSC development.

Inflammation fuels bone marrow exhaustion caused by Samd9l mutation.

Sterile α motif domain-containing 9 (SAMD9) and SAMD9-like (SAMD9L) syndromes are inherited bone marrow failure syndromes known for their frequent development of myelodysplastic syndrome with monosomy 7. In this issue of the JCI, Abdelhamed, Thomas, et al. report a mouse model with a hematopoietic cell-specific heterozygous Samd9l mutation knockin. This mouse model resembles human disease in many ways, including bone marrow failure and the nonrandom loss of the mutant allele. Samd9l-mutant hematopoietic stem progenitor cells showed reduced fitness at baseline, which was further exacerbated by inflammation. TGF-β hyperactivation was found to underlie reduced fitness, which was partially rescued by a TGF-β inhibitor. These findings illustrate the potential role of TGF-β inhibitors in the treatment of SAMD9/SAMD9L syndromes.

Thioredoxin-1 regulates self-renewal and differentiation of murine hematopoietic stem cells through p53 tumor suppressor

BackgroundThioredoxin-1 (TXN1) is one of the major cellular antioxidants in mammals and is involved in a wide range of physiological cellular responses. However, little is known about the roles and the underlying molecular mechanisms of TXN1 in the regulation of hematopoietic stem/progenitor cells (HSPCs).MethodsTXN1 conditional knockout mice (ROSA-CreER-TXN1fl/fl) and TXN1fl/fl control mice were used. The mice were treated with tamoxifen and the number and biological functions of HSPCs were measured by flow cytometry, PCR and western blot. Limiting dilution competitive transplantation with sorted HSCs and serial transplantations were performed to assess the effects of TXN1 knockout on HSC self-renewal and long-term reconstitutional capacity. RNA sequencing (RNA-seq) was performed to investigate the downstream molecular pathways of TXN1 deletion in murine HSPCs. CRISPR/Cas9 knockout experiments were performed in vitro in EML murine hematopoietic stem/progenitor cell line to investigate the effects of TXN1 and/or TP53 deletion on cell survival, senescence and colony forming units. TP53 protein degradation assay, CHiP PCR and PGL3 firefly/renilla reporter assay were performed. The effects of TXN1 on various molecular pathways relevant to HSC radiation protection were examined in vitro and in vivo.ResultsTXN1-TP53 tumor suppressor axis regulates HSPC biological fitness. Deletion of TXN1 in HSPCs using in vivo and in vitro models activates TP53 signaling pathway, and attenuates HSPC capacity to reconstitute hematopoiesis. Furthermore, we found that knocking out of TXN1 renders HSPCs more sensitive to radiation and treatment with recombinant TXN1 promotes the proliferation and expansion of HSPCs.ConclusionsOur findings suggest that TXN1-TP53 axis acts as a regulatory mechanism in HSPC biological functions. Additionally, our study demonstrates the clinical potential of TXN1 for enhancing hematopoietic recovery in hematopoietic stem cell transplant and protecting HSPCs from radiation injury.

Angelica sinensis polysaccharides prevents hematopoietic regression in D-Galactose-Induced aging model via attenuation of oxidative stress in hematopoietic microenvironment.

Extrinsic molecular mechanisms that regulate hematopoietic stem/progenitor cell (HSPC) aging are still poorly understood, and a potential protective medication needs to be explored. The senescent parameters of hematopoietic cells and bone marrow stromal cells (BMSCs) including cell cycle analysis, senescence-associated SA-β-gal staining and signals, hematopoietic factors and cellular junction were analyzed in femur and tibia of rats. Furthermore, Sca-1+ HSPCs and BMSCs co-culture system was established to evaluate the direct effects of BMSC feeder layer to HSPCs. Oxidative DNA damage indicators in Sca-1+ HSCs and senescence-associated secretory phenotype (SASP) of BMSCs, gap junction intercellular communication between BMSCs, osteogenesis/adipogenisis differentiation balance of BMSCs were detected. In the D-gal pre-administrated rats, ASP treatment rescued senescence of hematopoietic cells and BMSCs, reserved CFU-GEMM; also, ASP treatment attenuated stromal oxidative load, ameliorated SCF, CXCL12, and GM-CSF production, increased Connexin-43 (Cx43) expression. BMSCs and Sca-1+ HSPCs co-cultivation demonstrated that ASP treatment prevented oxidative DNA damage response in co-cultured Sca-1+ HSPCs induced by D-gal pre-administration of feeder layer and the underlying mechanism may be related to ASP ameliorating feeder layer dysfunction due to D-gal induced senescence via inhibiting secretion of IL-1, IL-6, TNF-α, and RANTES, enhancing Cx43-mediated intercellular communication, improving Runx2 expression whereas decreasing PPARγ expression in BMSCs. The antioxidant property of ASP may provide a stroma-mediated potential therapeutic strategy for HSPC aging.

Subclones of bone marrow CD34+ cells in acute myeloid leukemia at diagnosis confer responses of patients to induction chemotherapy.

Acute myeloid leukemia (AML) is a hematopoietic malignancy with a prognosis that varies with genetic heterogeneity of hematopoietic stem/progenitor cells (HSPCs). Induction chemotherapy with cytarabine and anthracycline has been the standard care for newly diagnosed AML, but about 30% of patients have no response to this regimen. The resistance mechanisms require deeper understanding. In our study, using single-cell RNA sequencing, we analyzed the heterogeneity of bone marrow CD34+ cells from newly diagnosed patients with AML who were then divided into sensitive and resistant groups according to their responses to induction chemotherapy with cytarabine and anthracycline. We verified our findings by TCGA database, GEO datasets, and multiparameter flow cytometry. We established a landscape for AML CD34+ cells and identified HSPC types based on the lineage signature genes. Interestingly, we found a cell population with CRIP1high LGALS1high S100Ashigh showing features of granulocyte-monocyte progenitors was associated with poor prognosis of AML. And two cell populations marked by CD34+ CD52+ or CD34+ CD74+ DAP12+ were related to good response to induction therapy, showing characteristics of hematopoietic stem cells. Our study indicates the subclones of CD34+ cells confers for outcomes of AML and provides biomarkers to predict the response of patients with AML to induction chemotherapy.

A novel BRET-Based GAP assay reveals phosphorylation-dependent regulation of the RAC-specific GTPase activating protein ARHGAP25.

ARHGAP25, a RAC-specific GTPase activating protein (GAP), is an essential regulator of phagocyte effector functions such as phagocytosis, superoxide production, and transendothelial migration. Furthermore, its complex role in tumor behavior has recently been recognized. We previously demonstrated that phosphorylation of serine 363 in ARHGAP25 regulates hematopoietic stem cells and progenitor cells in mouse bone marrow. However, the significance of other potential phosphorylation sites of ARHGAP25 remained unknown. Now, we developed a novel, real-time bioluminescence resonance energy transfer (BRET) assay to monitor the GAP activity of ARHGAP25 in vitro. Using this approach, we revealed that phosphorylation of S363 and S488, but not that of S379-380, controls ARHGAP25's RACGAP activity. On the other hand, we found in granulocyte-differentiated human PLB-985 cells that superoxide production and actin depolymerization are regulated by residues S363 and S379-380. The present data demonstrate the value of our BRET-GAP assay and show that different phosphorylation patterns regulate ARHGAP25's GAP activity and its effect on superoxide production and phagocytosis.