Normal Hematopoietic Stem Cells Research Articles

TGF-β1 protein trap AVID200 beneficially affects hematopoiesis and bone marrow fibrosis in myelofibrosis

Myelofibrosis (MF) is a progressive chronic myeloproliferative neoplasm characterized by hyperactivation of JAK/STAT signaling and dysregulation of the transcription factor GATA1 in megakaryocytes (MKs). TGF-β plays a pivotal role in the pathobiology of MF by promoting BM fibrosis and collagen deposition and by enhancing the dormancy of normal hematopoietic stem cells (HSCs). In this study, we show that MF-MKs elaborated significantly greater levels of TGF-β1 than TGF-β2 and TGF-β3 to a varying degree, and we evaluated the ability of AVID200, a potent TGF-β1/TGF-β3 protein trap, to block the excessive TGF-β signaling. Treatment of human mesenchymal stromal cells with AVID200 significantly reduced their proliferation, decreased phosphorylation of SMAD2, and interfered with the ability of TGF-β1 to induce collagen expression. Moreover, treatment of MF mononuclear cells with AVID200 led to increased numbers of progenitor cells (PCs) with WT JAK2 rather than mutated JAK2V617F. This effect of AVID200 on MF PCs was attributed to its ability to block TGF-β1–induced p57Kip2 expression and SMAD2 activation, thereby allowing normal rather than MF PCs to preferentially proliferate and form hematopoietic colonies. To assess the in vivo effects of AVID200, Gata1lo mice, a murine model of MF, were treated with AVID200, resulting in the reduction in BM fibrosis and an increase in BM cellularity. AVID200 treatment also increased the frequency and numbers of murine progenitor cells as well as short-term and long-term HSCs. Collectively, these data provide the rationale for TGF-β1 blockade, with AVID200 as a therapeutic strategy for patients with MF.

Hsa-circ_0003420 induces apoptosis in acute myeloid leukemia stem cells and impairs stem cell properties

Objective To explore the effect of circular RNA-0003420 (circ_0003420) in acute myeloid leukemia (AML) relapse and leukemia stem cells (LSCs) properties. Materials and methods TRIzol reagent was used to extract total RNA from AML tissues or cells. Cell viability was assessed by Cell Counting Kit-8 and EdU staining assays. Cell apoptosis was determined by flow cytometry. Results Compared with normal hematopoietic stem cells, circular RNA hsa-circ_0003420 expression was considerably decreased in non-m3 AML stem cells. Furthermore, the lack of hsa-circ_0003420 is correlated with poor clinical results and impaired therapeutic effects in AML. Overexpression of hsa-circ_0003420 via transfection caused LSC death and inhibited the characteristics of leukemia tumor stem cells, including expression of Homeobox B4 (HOXB4), MYB, and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) axis. Furthermore, hsa-circ_0003420 targets the mRNA of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) and hsa-circ_0003420 expression markedly repressed IGF2BP1 levels in LSCs. Restoration of IGF2BP1 eliminated the effect of hsa-circ_0003420 on the replication, apoptosis, and LSC phenotype of KG-1a cells. Discussion and conclusions Up-regulation of hsa-circ_0003420 expression in LSCs caused redox disorder, inflammation and apoptosis, suggesting that this protein could be used as a target for the treatment AML.

In This Issue

See article, p. 434.Ficlatuzumab is a first-in-class monoclonal antibody that blocks oncogenic MET signaling by blocking its ligand HGF. Here Wang et al. report on the safety and efficacy of ficlatuzumab combination with cytarabine in 17 patients with relapsed/refractory acute myeloid leukemia. Nine of the patients (53%) achieved a complete remission. Progression-free survival was 31.2 months, and median overall survival has not been reached. These outcomes compare favorably to historical data but remain to be validated in larger, randomized trials. Phosphoproteomic analysis identified S6 and MET phosphorylation as molecular correlates of response. Ras and GTPase gene expression signature was higher in prospective responders, whereas prospective nonresponders had elevated interferon and Myc signature.See article, p. 450.Human T-cell leukemia virus type-1 (HTLV-1) infection can progress to adult T-cell leukemia/lymphoma (ATL). To elucidate determinants of HTLV-1–driven oncogenesis, Koya et al. characterize circulating blood cells of healthy donors, HTLV-1 carriers, and patients with ATL by genome sequencing and CITE-seq combined with TCR-seq and BCR-seq. This high-dimensional single-cell analysis identifies Treg phenotype as a hallmark of premalignant clonal outgrowth. As the neoplastic clones progress to ATL, their expansion is accompanied by a proportional expansion of the myeloid compartment and immunosuppressive phenotypes including PD-L1 upregulation. The study delineates a new mechanism of PD-L1 acquisition by protein transfer from ATL to nonmalignant cells.See article, p. 468.Immunologic consequences of cell death contribute to clinical efficacy of certain chemotherapeutics, and identification of immunogenic drugs is essential for their optimal clinical use alone and in combination with immunotherapy. In this work, Gulla et al. have shown that a specific anti–multiple myeloma (MM) immunity occurs upon cell death induced by the proteasome inhibitor bortezomib, leading to immunologic memory and long-term clinical response. Intrinsic activation of STING followed by a viral mimicry state further contributes to the immunogenic effect, and combination of bortezomib with a STING agonist potentiates tumor cell–immune microenvironment interplay. These findings invite a deeper characterization of the immunologic effects of anti-MM agents and inform novel chemo-immunotherapy combinations.See article, p. 484.To obtain insight into the etiology of childhood leukemia, Brandsma et al. determined mutation burden of leukemic blasts and normal hematopoietic stem and progenitor cells (HSPC) from pediatric acute myeloid leukemia (pAML) patients and healthy age-matched controls. Integrating these data with available adult cohorts, they established a baseline of HSPC mutation accrual over the entire human lifespan. A subset of pediatric leukemia cases falling above the baseline harbored more genetic driver events and associated with better survival. The mutation pattern, characteristic of cells with high proliferation and oxidative stress, suggests a more committed progenitor as a cell of origin in this pAML subset.See article, p. 500.While the most frequent drivers of clonal hematopoiesis have been characterized previously, mutations in these genes are not present in about half of clonal hematopoiesis cases. By identifying recurrent mutations in the blood of large numbers of healthy people, Beauchamp et al. discover additional genes likely to drive clonal hematopoiesis and thus regulating hematopoietic stem cell (HSC) expansion. Modeling ZBTB33 mutations in mouse HSCs, the authors observe that the edited cells expand and have a competitive advantage compared with control cells. Cells with Zbtb33 mutations also have altered intron retention, potentially connecting ZBTB33 to RNA splicing, a commonly mutated pathway in clonal hematopoiesis and myelodysplastic syndromes.See article, p. 518.Romine et al. characterize acute myeloid leukemia (AML) sensitivity to BET inhibitors (BETi) by two orthogonal approaches: ex vivo drug screening of genetically defined primary AML samples, and genome-wide CRISPR screens of BETi-sensitive and BETi-resistant cells. Both identify loss of monocytic differentiation regulators as drivers of BETi resistance. BETi-resistant AML cells express low levels of myeloid markers; conversely, forced myeloid differentiation sensitizes AML cells to BETi. CRISPR dropout screens in BETi-resistant cells reveal BCL2 and CDK4/6 as targetable vulnerabilities to overcome BETi resistance. Moreover, monocytic AML refractory to BCL2 inhibition, an unmet clinical challenge, shows preclinical responses to combined Bcl2 and BETi.See article, p. 532.Binding of tyrosine kinase inhibitors (TKI) by plasma proteins, most notably alpha-1-acid glycoprotein (AGP), can dramatically reduce TKI bioavailability and thus clinical potency. In this work, Young et al. characterize AGP impact on staurosporine-derived TKI (STS-TKI) activity against FLT3-mutated acute myeloid leukemia (AML). Under physiologic conditions and drug concentrations, AGP quenches STS-TKI activity to subtherapeutic levels due to high-affinity drug–protein interactions. To overcome this, the authors screen for drugs restoring TKI activity in the presence of AGP, identifying mifepristone. In an in vivo model, mifepristone coadministration restores midostaurin clearance of AML cells. These results suggest a combinatorial approach for unlocking TKI activity to potentiate therapeutic responses in FLT3-mutated AML.

ALL-015: Stem Cells in Acute Lymphoblastic Leukemia and In Vitro Assessment of the Effect of Parthenolide

Introduction: In ALL, current drug efficacy studies focus on reducing leukemia cell burden. However, if drugs have limited effects on LSCs, these cells may expand and eventually cause relapse. The experimental anti-leukemic drug parthenolide (PTL) acts by inhibiting transcription factor nuclear kappa B ( NFκB ) , activating p53 and increasing reactive oxygen species (ROS) in leukemic cells. In the present study we assessed the in vitro effect of PTL on immune-phenotypically defined leukemic stem cells (LSCs) and normal hematopoietic stem cells (HSCs). Method: Forty ALL samples and 24 normal bone marrow samples were included in this study. ALL samples were sorted into 4 LSC populations (CD34+CD38-CD19+, CD34+CD38+CD19+, CD34+CD38-CD19-, and CD34-CD38-CD19-). Cells were cultured with different PTL concentrations for 24, 48 and 72 hours. Post-culture cell viability was assessed using 7ADD in a flow cytometry-based test. Normal unsorted marrow cells were tested under similar conditions. In addition, colony forming unit assay (CFU) was carried out to assess the effect of PTL on HSCs. Results: In ALL, LSCs form heterogeneous compartments with different percentages of the four LSC populations. This heterogeneity extended across and within cytogenetically classified groups of cases. There was no significant effect of PTL on the viability of normal cells at concentrations of ≤ 50μΜ; but at 25 μΜ and 10 μΜ after 24 and 72 hours respectively it affected the ability of HSCs to form colonies. For ALL-LSCs PTL was significantly toxic at10 μΜ and most cases with the exception of those with +3 karyotype abnormality and CD34-CD38-CD19- LSCs with t(9;22), showed >50% cell-death at 25μΜ PTL concentration. The CD34+, CD38+, CD19+ LSCs showed significantly higher sensitivity compared to other subpopulations. ALL cases with limited response to PTL expressed significantly higher levels of pNFκBP65 as compared to PTL-sensitive cases. Conclusion: Overall, CD34+CD38-CD19+ and CD34+CD38-CD19 cells are the predominant LSC populations in B-ALL. PTL has a differential effect on normal HSCs versus ALL cells at lower concentrations in vitro. PTL also has a differential effect on LSC subpopulations. PTL sensitivity/resistance in ALL is related to the level of activated NFκB expression in these cells.

MDS-420: Sabatolimab Plus Hypomethylating Agents (HMAs) in Patients with High-/Very High-risk Myelodysplastic Syndrome (HR/vHR-MDS) and Newly Diagnosed Acute Myeloid Leukemia (ND-AML): Subgroup Analysis of a Phase 1 Study

Context: Sabatolimab (MBG453) is an investigational immuno-myeloid therapy targeting TIM-3, an immune regulator expressed on immune and myeloid leukemic cells and blasts, but not on normal hematopoietic stem cells. Early phase 1b study (NCT03066648) results showed sabatolimab+HMA treatment had promising and durable activity in MDS/AML. Objective: To further explore safety/tolerability and efficacy in patient subgroups, as well as biomarkers with sabatolimab+HMA. Design: Study design/eligibility criteria have been reported. HMA-naive patients with HR/vHR-MDS and ND-AML and ineligible for intensive chemotherapy received sabatolimab (1–2 infusions/month) + decitabine or azacitidine. Primary objectives: safety/tolerability. Secondary objectives: PK and preliminary efficacy. Data cutoff was 22 September 2020; updated data will be presented. Results: Of 89 patients with HR/vHR-MDS (n=41) and ND-AML (n=48), 36 (40%) had sabatolimab dose interruption, 1 (1%) had dose reduction, 2 (2%) had dose interruption and reduction, 5 (6%) discontinued, and 3 (3%) had dose interruption and discontinued. Four discontinuations were due to AEs and 4 due to death. Twenty-two patients had grade 4 neutropenia/thrombocytopenia at baseline: 4 (18%) had dose interruption, 2 (9%) discontinued, and 1 (5%) had dose interruption and discontinued. In analyses of remission rates (CR+mCR/CRi+PR) by baseline factors, response was independent of bone marrow blast burden in patients with HR/vHR-MDS or ND-AML. Remission rates were similar in patients age ≥75 and 65–74 years: 50% (6/12) and 65% (11/17) with HR/vHR-MDS and 42% (8/19) for both groups with ND-AML. Response durability in patients age ≥75 and 65–74 years was encouraging: an estimated 83% and 86%, respectively, with HR/vHR-MDS remained in remission after 6 months. In patients with TP53 mutation or ≥1 mutation conferring European LeukemiaNet (ELN) high risk, remission rates were 55% (6/11; 4/6 in remission >200 days) and 59% (13/22; 8/13 in remission >200 days), respectively, for HR/vHR-MDS. Durability and remission rates for ND-AML will be presented. Biomarker analyses identified differential expression of IL-1β in sabatolimab+HMA responders vs nonresponders; expression levels were inversely correlated with remission. Conclusions: Sabatolimab+HMA showed favorable tolerability in MDS/AML, including in patients with grade 4 cytopenias at baseline. Promising remission rates were observed across patient subgroups. This supports development of sabatolimab+HMA in the STIMULUS trial program of MDS/AML.

PI3K/mTOR dual-inhibition with VS-5584 enhances anti-leukemic efficacy of ponatinib in blasts and Ph-negative LSCs of chronic myeloid leukemia

Ponatinib is used for advanced treatment of chronic myeloid leukemia (CML), although low doses to prevent side effects do not suppress survival pathways and eradicate leukemia stem cells (LSCs). We evaluated the potential of ponatinib and PI3K/mTOR dual-inhibitor VS-5584 combination (PoVS) therapy to increase the anti-leukemic effects of ponatinib and investigated the underlying mechanisms at the molecular level. We measured the cytotoxicities of ponatinib, VS-5584, and PoVS (CCK-8 assay), and used the median-effect equation for combination analyses. We investigated the effects of inhibitory concentrations on apoptosis, cell viability and cell-cycle regulation (flow cytometry), protein levels (ELISA, Western blot), transcriptional activities (dual-luciferase reporter assay), gene expressions (qRT-PCR). VS-5584 exerted selective cytotoxic effects against CML and LSC cell lines. VS-5584 inhibited the PI3K/Akt/mTOR pathway, resulting in reduced cell viability, slightly induced caspase-independent apoptosis, prominent G0/G1 cell-cycle blockade that is not a consequence of quiescence. Normal hematopoietic stem cell line was the least affected. Moreover, ponatinib and VS-5584 mediated synergistic anti-leukemic effects on leukemic cells. VS-5584 reduced the ponatinib dose required to target leukemic cells. PoVS treatment inhibited PI3K/Akt/mTOR pathway more consistently than either of the two agents alone through reducing p-Akt, p-mTOR, p-S6K, p-PRAS40, p-S6. The subsequent downstream effects were an increase in C/EBP transcriptional activity and decreases in activities of E2F/DP1, Myc/Max, CREB, STAT3, NFκB, AP-1, Elk-1/SRF. Transcriptional regulation resulted in alterations in the expression levels of target mRNAs. Our results highlight PoVS can be a promising treatment strategy for eliminating CML cells and LSCs selectively, with the reduced ponatinib doses.

Therapeutic Targeting of Mesothelin with Chimeric Antigen Receptor T Cells in Acute Myeloid Leukemia.

We previously identified mesothelin (MSLN) as highly expressed in a significant fraction of acute myeloid leukemia (AML) but entirely silent in normal hematopoiesis, providing a promising antigen for immunotherapeutic targeting that avoids hematopoietic toxicity. Given that T cells genetically modified to express chimeric antigen receptors (CAR) are effective at eradicating relapsed/refractory acute lymphocytic leukemia, we developed MSLN-directed CAR T cells for preclinical evaluation in AML. The variable light (VL) and heavy (VH) sequences from the MSLN-targeting SS1P immunotoxin were used to construct the single-chain variable fragment of the standard CAR containing 41-BB costimulatory and CD3Zeta stimulatory domains. The preclinical efficacy of MSLN CAR T cells was evaluated against AML cell lines and patient samples expressing various levels of MSLN in vitro and in vivo. We demonstrate that MSLN is expressed on the cell surface of AML blasts and leukemic stem cell-enriched CD34+CD38- subset, but not on normal hematopoietic stem and progenitor cells (HSPC). We further establish that MSLN CAR T cells are highly effective in eliminating MSLN-positive AML cells in cell line- and patient-derived xenograft models. Importantly, MSLN CAR T cells can target and eradicate CD34+CD38- cells without impacting the viability of normal HSPCs. Finally, we show that CAR T-cell functionality can be improved by inhibition of the ADAM17 metalloprotease that promotes shedding of MSLN. These findings demonstrate that MSLN is a viable target for CAR T-cell therapy in AML and that inhibiting MSLN shedding is a promising approach to improve CAR T-cell efficacy.

Expanding the immune armoury against myelodysplastic syndrome.

Expanding the immune armoury against myelodysplastic syndrome.

RSPO2 inhibits BMP signaling to promote self-renewal in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a rapidly progressing cancer, for which chemotherapy remains standard treatment and additional therapeutic targets are requisite. Here, we show that AML cells secrete the stem cell growth factor R-spondin 2 (RSPO2) to promote their self-renewal and prevent cell differentiation. Although RSPO2 is a well-known WNT agonist, we reveal that it maintains AML self-renewal WNT independently, by inhibiting BMP receptor signaling. Autocrine RSPO2 signaling is also required to prevent differentiation and to promote self-renewal in normal hematopoietic stem cells as well as primary AML cells. Comprehensive datamining reveals that RSPO2 expression is elevated in patients with AML of poor prognosis. Consistently, inhibiting RSPO2 prolongs survival in AML mouse xenograft models. Our study indicates that in AML, RSPO2 acts as an autocrine BMP antagonist to promote cancer cell renewal and may serve as a marker for poor prognosis.

Infection makes micro-CHIPs into macro-CHIPs.

In this issue of Cell Stem Cell, Hormaechea-Agulla etal. (2021) demonstrate that IFNγ signaling following an infection in mice provides a selective pressure that drives growth of Dnmt3a-/- hematopoietic stem cells. This clonal expansion is mediated by global methylation changes that lead to an increased self-renewing capacity.

Transcription Factors, R-Loops and Deubiquitinating Enzymes: Emerging Targets in Myelodysplastic Syndromes and Acute Myeloid Leukemia.

Simple SummaryThe advent of DNA massive sequencing technologies has allowed for the first time an extensive look into the heterogeneous spectrum of genes and mutations underpinning myelodysplastic syndromes (MDSs) and acute myeloid leukemia (AML). In this review, we wish to explore the most recent advances and the rationale for the potential therapeutic interest of three main actors in myelo-leukemic transformation: transcription factors that govern myeloid differentiation; RNA splicing factors, which ensure proper mRNA maturation and whose mutations increase R-loops formation; and deubiquitinating enzymes, which contribute to genome stability in hematopoietic stem cells (HSCs).Myeloid neoplasms encompass a very heterogeneous family of diseases characterized by the failure of the molecular mechanisms that ensure a balanced equilibrium between hematopoietic stem cells (HSCs) self-renewal and the proper production of differentiated cells. The origin of the driver mutations leading to preleukemia can be traced back to HSC/progenitor cells. Many properties typical to normal HSCs are exploited by leukemic stem cells (LSCs) to their advantage, leading to the emergence of a clonal population that can eventually progress to leukemia with variable latency and evolution. In fact, different subclones might in turn develop from the original malignant clone through accumulation of additional mutations, increasing their competitive fitness. This process ultimately leads to a complex cancer architecture where a mosaic of cellular clones—each carrying a unique set of mutations—coexists. The repertoire of genes whose mutations contribute to the progression toward leukemogenesis is broad. It encompasses genes involved in different cellular processes, including transcriptional regulation, epigenetics (DNA and histones modifications), DNA damage signaling and repair, chromosome segregation and replication (cohesin complex), RNA splicing, and signal transduction. Among these many players, transcription factors, RNA splicing proteins, and deubiquitinating enzymes are emerging as potential targets for therapeutic intervention.

Siglec-6 is a novel target for CAR T-cell therapy in acute myeloid leukemia

AbstractAcute myeloid leukemia (AML) is an attractive entity for the development of chimeric antigen receptor (CAR) T-cell immunotherapy because AML blasts are susceptible to T-cell–mediated elimination. Here, we introduce sialic acid–binding immunoglobulin-like lectin 6 (Siglec-6) as a novel target for CAR T cells in AML. We designed a Siglec-6–specific CAR with a targeting domain derived from the human monoclonal antibody JML-1. We found that Siglec-6 is commonly expressed on AML cell lines and primary AML blasts, including the subpopulation of AML stem cells. Treatment with Siglec-6 CAR T cells confers specific antileukemia reactivity that correlates with Siglec-6 expression in preclinical models, including induction of complete remission in a xenograft AML model in immunodeficient mice (NSG/U937). In addition, we confirmed Siglec-6 expression on transformed B cells in chronic lymphocytic leukemia (CLL), and specific anti-CLL reactivity of Siglec-6 CAR T cells in vitro. Of particular interest, we found that Siglec-6 is not detectable on normal hematopoietic stem and progenitor cells (HSPCs) and that treatment with Siglec-6 CAR T cells does not affect their viability and lineage differentiation in colony-formation assays. These data suggest that Siglec-6 CAR T-cell therapy may be used to effectively treat AML without the need for subsequent allogeneic hematopoietic stem cell transplantation. In mature normal hematopoietic cells, we detected Siglec-6 in a proportion of memory (and naïve) B cells and basophilic granulocytes, suggesting the potential for limited on-target/off-tumor reactivity. The lack of expression of Siglec-6 on normal HSPCs is a key to differentiating it from other Siglec family members (eg, Siglec-3 [CD33]) and other CAR target antigens (eg, CD123) that are under investigation in AML, and it warrants the clinical investigation of Siglec-6 CAR T-cell therapy.

Stem Cells in the Myelodysplastic Syndromes.

The myelodysplastic syndromes (MDS) represent a group of clonal disorders characterized by ineffective hematopoiesis, resulting in peripheral cytopenias and frequent transformation to acute myeloid leukemia (AML). We and others have demonstrated that MDS arises in, and is propagated by malignant stem cells (MDS-SCs), that arise due to the sequential acquisition of genetic and epigenetic alterations in normal hematopoietic stem cells (HSCs). This review focuses on recent advancements in the cellular and molecular characterization of MDS-SCs, as well as their role in mediating MDS clinical outcomes. In addition to discussing the cell surface proteins aberrantly upregulated on MDS-SCs that have allowed the identification and prospective isolation of MDS-SCs, we will discuss the recurrent cytogenetic abnormalities and genetic mutations present in MDS-SCs and their roles in initiating disease, including recent studies demonstrating patterns of clonal evolution and disease progression from pre-malignant HSCs to MDS-SCs. We also will discuss the pathways that have been described as drivers or promoters of disease, including hyperactivated innate immune signaling, and how the identification of these alterations in MDS-SC have led to investigations of novel therapeutic strategies to treat MDS. It is important to note that despite our increasing understanding of the pathogenesis of MDS, the molecular mechanisms that drive responses to therapy remain poorly understood, especially the mechanisms that underlie and distinguish hematologic improvement from reductions in blast burden. Ultimately, such distinctions will be required in order to determine the shared and/or unique molecular mechanisms that drive ineffective hematopoiesis, MDS-SC maintenance, and leukemic transformation.

A critical role of nuclear m6A reader YTHDC1 in leukemogenesis by regulating MCM complex–mediated DNA replication

AbstractYTHDC1 has distinct functions as a nuclear N6-methyladenosine (m6A) reader in regulating RNA metabolism. Here we show that YTHDC1 is overexpressed in acute myeloid leukemia (AML) and that it is required for the proliferation and survival of human AML cells. Genetic deletion of Ythdc1 markedly blocks AML development and maintenance as well as self-renewal of leukemia stem cells (LSCs) in vivo in mice. We found that Ythdc1 is also required for normal hematopoiesis and hematopoietic stem and progenitor cell (HSPC) maintenance in vivo. Notably, Ythdc1 haploinsufficiency reduces self-renewal of LSCs but not HSPCs in vivo. YTHDC1 knockdown has a strong inhibitory effect on proliferation of primary AML cells. Mechanistically, YTHDC1 regulates leukemogenesis through MCM4, which is a critical regulator of DNA replication. Our study provides compelling evidence that shows an oncogenic role and a distinct mechanism of YTHDC1 in AML.

TIM-3 in normal and malignant hematopoiesis: Structure, function, and signaling pathways.

Acute myeloid leukemia (AML) is hierarchically organized by self‐renewing leukemic stem cells (LSCs). LSCs originate from hematopoietic stem cells (HSCs) by acquiring multistep leukemogenic events. To specifically eradicate LSCs, while keeping normal HSCs intact, the discrimination of LSCs from HSCs is important. We have identified T‐cell immunoglobulin and mucin‐domain containing‐3 (TIM‐3) as an LSC‐specific surface molecule in human myeloid malignancies and demonstrated its essential function in maintaining the self‐renewal ability of LSCs. TIM‐3 has been intensively investigated as a “coinhibitory” or “immune checkpoint” molecule of T cells. However, little is known about its distinct function in T cells and myeloid malignancies. In this review, we discuss the structure of TIM‐3 and its function in normal blood cells and LSCs, emphasizing the specific signaling pathways involved, as well as the therapeutic applications of TIM‐3 molecules in human myeloid malignancies.

Abstract LB189: Three-dimensional genome organization maps in normal haematopoietic stem cells and acute myeloid leukemia

Abstract Many oncogenes, such as MEIS1 and HOXA9, are overexpressed in some but not all cancers. We identified two key epigenetic mechanisms underlying this heterogeneity in oncogene expression in Acute Myeloid Leukemia. Acute Myeloid Leukemia (AML) is a highly lethal blood cancer arising due to aberrant differentiation of haematopoietic stem cells. MEIS1 and HOXA9 regulate stemness-related transcriptional programs in normal haematopoietic stem cells and AML. Here we obtained 3D genome organization maps in the CD34+ haematopoietic stem cells from healthy individuals and individuals with AML. The MEIS1 oncogenic transcription factor is regulated by a Frequently Interacting Region (FIRE). This FIRE is present in normal bone marrow samples, and an AML sample with high MEIS1 levels. However, it is absent in two AML samples that show low MEIS1 levels. CRISPR excision of the FIRE led to loss of MEIS1 and reduced cell growth. Moreover, MEIS1 can bind to the promoter of HOXA9. HOXA9 can also auto-regulate by binding to its own promoter as well as an Acute Myeloid Leukemia-specific super-enhancer that interacts with the HOXA9 promoter via chromatin interactions. The mechanisms elucidated here could be potentially exploited to utilize epigenetic inhibitors to specifically target oncogene expression in cancer. Citation Format: Benny Wang, Lingshi Kong, Deepak Babu, Ruchi Choudhary, Winnie Fam, Jia Qi Tng, Yufen Goh, Xin Liu, Fang Fang Song, Priscella Chia, Ming Chun Chan, Omer An, Cheng Yong Tham, Touati Benoukraf, Henry Yang, Wilson Wang, Wee Joo Chng, Daniel Tenen, Melissa J. Fullwood. Three-dimensional genome organization maps in normal haematopoietic stem cells and acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB189.

Abstract 3099: The metabolic enzyme hexokinase 2 localizes to the nucleus in AML and normal hematopoietic stem/progenitor cells to maintain stemness

Abstract Mitochondrial metabolites affect epigenetic marks, but it is largely unknown whether mitochondrial metabolic enzymes can directly localize to the nucleus to regulate stem cell function in AML. Here, we discovered that the mitochondrial enzyme, Hexokinase 2 (HK2), localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stem cell function. We searched for mitochondrial enzymes moonlighting in the nucleus using 8227 AML cells, a primary AML culture model arranged in a hierarchy with defined stem cells. By immunoblotting and confocal microscopy, we detected HK2 in the nucleus of 8227 cells with higher expression in the nucleus of stem cells vs bulk cells. In contrast, other metabolic enzymes including PFK1, FH, PKM2, GPI1, ENO1, CS, ACO2, and SDHA1, 2 were not detected in the nucleus of these cells. We also detected HK2 in the nucleus of AML cell lines as well as 7 of 9 primary AML samples. Next, we tested whether nuclear HK2 was functionally important to maintain stem cell function in AML. We over-expressed HK2 tagged with nuclear localizing signals in 8227 and NB4 leukemia cells. This increased clonogenic growth and inhibited retinoic acid-mediated cell differentiation without changing basal proliferation. Nuclear HK2 also increased engraftment of 8227 cells into mouse marrow. We evaluated the selective inhibition of nuclear HK2 by over-expressing HK2 with an outer mitochondrial localization signal while knocking down endogenous HK2 with 3'UTR shRNA. Selective depletion of nuclear HK2 reduced clonogenic growth, increased AML differentiation with ATRA, and decreased CD34+CD38- 8227 stem cells without changing basal proliferation. Nuclear HK2 was also higher in normal human hematopoietic stem cells and multipotent progenitor fractions and declined as the cells matured. Over-expression of nuclear HK2 in normal cord blood increased the primary and secondary engraftment into mice. Transgenic mice over-expressing nuclear HK2 driven by a Vav promoter had increased hematopoietic stem cells in the marrow and decreased monocytes and lymphocytes in the peripheral blood. To determine whether nuclear HK2 maintains stemness through its kinase activity, we over-expressed a kinase dead double mutant of nuclear HK2 (D209A D657A) and observed increased clonogenic growth and inhibited differentiation on ATRA treatment, nuclear HK2 function is independent of its kinase function. To understand nuclear functions of HK2, we used proximity-dependent biotin labeling (BioID) and mass spectrometry identified proteins related to chromatin organization and regulation to interact with nuclear HK2. In summary, we discovered that HK2 localizes to nucleus of AML cells and functions independent of its kinase activity to maintain the stem/progenitor state of AML. Thus, we define a new role for mitochondrial enzymes in the regulation of leukemic stemness and differentiation. Citation Format: Geethu Emily Thomas, Grace Egan, Laura Gracia Prat, Aaron Botham, Veronique Voisin, Elias Orouji, Jordan Chin, Boaz Nachmias, Kerstin B. Kaufmann, Neil Maclean, Rose Hurren, Marcela Gronda, Xiaoming Wang, Dilshad H. Khan, Rashim P. Singh, Andrea Arruda, Mark Minden, Gary D. Bader, John E. Dick, Aaron D. Schimmer. The metabolic enzyme hexokinase 2 localizes to the nucleus in AML and normal hematopoietic stem/progenitor cells to maintain stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3099.

CXCL12-Expressing Bone Marrow Niches Differentially Regulate CML Compared to Normal Stem Cells

CXCL12 plays a major role in hematopoietic stem cells (HSC) localization to regulatory niches in the bone marrow microenvironment (BMM). Previous studies indicate that CXCL12 deletion from Prx+ mesenchymal stem cells (MSC) reduces murine HSC numbers, and from Tek+ endothelial cells (EC) modestly reduces their repopulating activity. In contrast, CXCL12 deletion from Ocn+ osteoblasts or Osx+ osteoprogenitors does not affect HSC function (Greenbaum et al. Nature. 2013 Mar 14;495(7440):227-30; Ding et al. Nature. 2013 Mar 14;495(7440):231-5.). These studies identified MSC and endothelial cells as important HSC niche components. However, niche cells for CML leukemic stem cells (LSC) remain poorly characterized. To evaluate the contribution of CXCL12-expressing populations to LSC regulation, we transplanted normal and CML BM cells (CD45.1/2+; 2*106/mouse) into CXCL12f/f (loxP sites flanking exon 2) mice crossed with Tek-Cre and Prx-Cre mice ( reported in ASH 2016, Blood, Abstract: 26) . We showed that CXCL12 deletion from Prx+ MSC resulted in reduction in normal HSC (LSK Flt3- CD150+ CD48- cells) numbers and function compared to controls, but did not observe significant reduction of HSC with CXCL12 deletion from Tek+ EC. Interestingly, transplantation of CML cells into mice with CXCL12 deleted from Prx+ MSC led to reduced survival compared to control mice associated with increased WBC counts, BM cellularity, LSC numbers, and secondary repopulation capacity. In contrast, CXCL12 deletion from Tek+ cells led to enhanced survival, associated with reduced BM cellularity and reduced LSC numbers. In further studies, we transplanted mixtures of equal numbers of normal and CML cells into Prx-Cre and Tek-Cre mice, and found a marked growth advantage of LSC compared with HSC in Prx-Cre mice, and a growth advantage of HSC over LSC in Tek-Cre knockout mice. We also show that both HSC and LSC demonstrate increased cell cycling via EdU labeling. Consistent with this, we found that Nilotinib treatment leads to significantly greater reduction in BM cellularity, LSC numbers and secondary repopulation potential in Prx-Cre mice, compared to Tek-Cre or WT mice. To evaluate the effects of CML on CXCL12-expressing BMM cells, we crossed CXCL12GFP (GFP reporter knocked into the CXCL12 locus) and SCL-tTA-BCR-ABL mice to generate CXCL12GFP-BCR-ABL mice. CML development lead to significantly reduced numbers of GFP+ BM stromal cells (CD45-Ter119-31-) but increased GFP+ endothelial cells (CD45-Ter119-CD31+), and within the stromal population, the number of GFP+ MSC (PDGFRα+Sca-1+) were decreased compared to WT mice. To further understand niche mediated interactions that regulate LSC fate, we studied gene expression profiles of LSC from mice following Cre-mediated targeting of CXCL12, compared to LSC from Cre-negative mice. Our preliminary analysis indicates significant enrichment of genes associated with cell cycle progression and Myc activation, and negative enrichment of genes related to TGF-b, STAT3, and Polycomb Repressive Complex 2 (PRC2) activity in LSC from Prx1+Cre CXCL12f/f mice. Reduced TGF-b, STAT3 and PRC2/EZH2 activity represent potential mechanisms for regulation of CML LSC maintenance and/or TKI resistance, and may potentially contribute to Prx1+ MSC-mediated LSC regulation. We conclude that CXCL12+ BM MSC inhibit CML development, maintain LSC quiescence, and enhance TKI resistance, while CXCL12+ EC enhance LSC maintenance and disease development. Leukemia associated reduction in CXCL12+MSC and increase in CXCL12+EC may enhance CML development. Collectively, these results reveal important and distinct niche functions for CXCL12 expressing BM MSC and EC for CML LSC compared with normal HSC. DisclosuresNo relevant conflicts of interest to declare.

Targeting the Epigenetic and Metabolic Vulnerabilities of Ezh2-Deficiency-Induced Myeloid Neoplasms

Myeloproliferative neoplasms (MPNs) are clonal, progressive hematopoietic malignancies characterized by alterations of multiple signaling and epigenetic pathways in hematopoietic stem cells (HSCs). The identification of driver oncogenic mutations in JAK2, CALR or MPL has transformed our knowledge of MPNs; however, patients with non-mutated JAK2, CALR and MPL (so-called ‘triple-negative‘) have the highest incidence of leukemic transformation and the lowest overall survival (Rumi et al., 2014 Blood 124:1062-1069), indicating that other factors may contribute to MPNs. The molecular processes controlling MPN progression from chronic to acute leukemic transformation remain unknown. This is a major impediment for developing target-based therapeutics to selectively eliminate the mutant stem cells to prevent disease progression and/or relapse. Therefore, the challenge is twofold: 1) to identify and characterize the genetic complexity of MPN pathophysiology, and 2) to understand and control the progression of MPNs from chronic to life-threatening stages.We have developed a new mouse model of MPNs by hematopoietic-selective (Mx1-cre) activation of oncogenic Ras (NRasG12D+/-) and inactivation of Ezh2, the enzymatic subunit of the Polycomb Repressive Complex 2 (PRC2), which mimics concurrent mutations in human MPNs. While NRasG12D+/- alone led to an indolent myeloproliferative disorder, Ezh2 loss markedly accelerated disease progression to life-threatening stages, resulting in thrombocytosis, neutrophilia, splenomegaly, primary myelofibrosis, leukemic transformation and mortality. Strikingly, while loss of Ezh1, a homolog of Ezh2, had no effect on normal hematopoiesis, concurrent inactivation of Ezh1 and Ezh2 with NRasG12D+/- completely abolished MPN development and myelofibrosis in vivo . These results establish distinct functions of Ezh1 and Ezh2 in myeloid neoplasms, and identify a selective epigenetic vulnerability for MPNs induced by Ezh2-deficiency. Follow-up studies of Ezh1/2-mediated chromatin (by ChIP-seq) and transcriptomic (by RNA-seq) changes in normal HSCs and leukemia-initiating cells (LICs) revealed non-overlapping gene targets and cellular pathways required for the distinct cellular phenotypes.Reprogrammed cellular metabolism underlies the progression of various human cancers including leukemia. We noted by RNA-seq transcriptomic profiling that Bcat1, the first and rate-limiting enzyme catalyzing the transamination of branched-chain amino acids (BCAAs), was aberrantly activated in Ezh2-deficient leukemia-initiating cells (LICs). By ChIP-seq analysis, we observed that the Bcat1 promoter is highly enriched with tri-methylated H3-Lys27 (H3K27me3), suggesting that it is epigenetically silenced by Ezh2-PRC2 in normal HSCs. Furthermore, genetic or pharmacologic inhibition of BCAT1 activity selectively abrogated the clonogenic activity of Ezh2-deficient LICs in vitro and the leukemogenic potential in transplanted recipients in vivo, highlighting a metabolic dependency for Ezh2-mutant myeloid neoplasms. Follow-up targeted metabolomic analysis of Ezh2-deficiency-induced metabolic changes established the critical role of Bcat1 and the associated BCAA metabolism in the progression of myeloid neoplasms and leukemia.Collectively, our results not only elucidate the mechanisms by which Ezh1 is specifically required for Ezh2-deficient LICs, but also validate an important epigenetic vulnerability for MPN patients carrying loss-of-function Ezh2 mutations. Moreover, in-depth analysis of BCAA metabolism in myeloid neoplasms identifies a new metabolic dependency for Ezh2-deficient LICs. Because normal hematopoietic stem cells are unaffected by loss of Ezh1 or Bcat1, the selective vulnerabilities raise the possibility of leveraging Ezh1 or BCAA metabolism as targeted therapies to specifically eradicate Ezh2-mutant LICs. Hence, our study promises to provide critical insights into developing new and generalizable therapeutic strategies to selectively eliminate leukemia-initiating cells harboring alterations of epigenetic pathways. DisclosuresDeberardinis:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Two Distinct but Functionally Related Autoreactive T Cell Populations in Paroxysmal Nocturnal Hemoglobinuria and Idiopathic Aplastic Anemia: Glycosylphosphatidylinositol-Specific and IFNγ-Producing T Cells

Paroxysmal nocturnal hemoglobinuria (PNH) and idiopathic aplastic anemia (IAA) are closely related disorders: several lines of evidences indicate that both have a T-cell-mediated autoimmune pathogenesis.We have previously reported that in almost all PNH patients, compared to controls, there is a significant increase of CD8 T cells (CDd1-restricted, GPI-specific T cells), that through their TCR recognize a CD1d dimer loaded with glycosylphosphatidylinositol (GPI); as well as of CD8 T cells producing interferon gamma (IFNγ) in response to GPI presented by CD1d (CDd1-restricted, GPI-reactive T cells). Recently, we have found that GPI specific T cells were also present in about 70% of IAA patients and that only in this subset of AA patients there was also an increased frequency of INFγ producing T cells upon GPI presentation. We have postulated that, through the toxic effect exerted by IFNγ upon the TCR recognition of GPI within the CD1d groove, these cells target selectively normal (GPI-positive) hematopoietic stem cells (HSC), whereas the PNH (GPI-negative) HSC are spared.Here we first confirm in a new series of 14 PNH patients that the frequency of GPI-specific and GPI reactive T cells increase when peripheral blood mononuclear cells (PBMNC) from PNH patients are grown on antigen presenting cells (APC) expressing CD1d, with the condition that either endogenous or exogenous GPI Is present We have also confirmed that this increase is restricted to CD8 T cells. Next, we have characterized these cells after 7 days of co-culture: we find that GPI specific T cells (CD8+CD1d/GPI dimer+ T cells) and INFγ producing T cells (CD8+INFγ+ T cells) did not express any of the following surface molecules: CD25, IL17, CD56 and CD16.In all our experiments, INFγ producing T cells outnumber GPI-specific T cells: suggesting that INFγ response is not restricted to GPI-specific T cells (those that are able to recognize the GPI presented by CD1d APCs). In order to dissect the relationship between the two cell types thus defined, after 7 days of co-culture with CD1d APCs competent for GPI synthesis, we have performed simultaneous staining with both GPI-loaded CD1d-dimer and anti-IFNγ. Surprisingly, we found that in none of 20 patients (14 PNH and 6 IAA) were present CD8 T cells stained with both GPI-loaded CD1d-dimer and intracellular anti-IFNγ. CD1d/GPI dimer+ T cells were 0.8±0.32%; INFγ+ T cells were 3.22±0.56%; CD1d/GPI dimer+/INFγ+ T cells were 0.04±0.01% (Fig. 1A). Thus, the T cells that increase specifically after the CD1d mediated GPI presentation consist of two distinct populations. Since their frequency increases in response to GPI stimulus, we surmised that the GPI-specific T cells upon TCR engagement with the GPI loaded CD1d activate in a specific manner a population of CD8 T cell that produce IFNγ. We have tested this hypothesis by comparing in the same setting (co-culture on CD1d APCs either unable or able to synthesize GPI) the PBMNC of 8 PNH patients with and without depletion by immunomagnetic sorting of T cells able to bind the CD1d/GPI dimer: without depletion we observed, as expected, an increased frequency of both GPI-specific and of IFNγ producing T cells; in contrast, with CD1d/dimer+ depleted PBMNC, we observed not only the absence of GPI-specific T cells (confirming that the depletion procedure had been effective), but also a marked reduction of INFγ producing (GPI reactive) T cells (Fig. 1B).This study indicates that both GPI specific (CD8+ CD1d/GPI dimer+) T cells and INFγ producing (CD8+INFγ+) T cells increase specifically after CD1d-mediated GPI presentation. At the moment we do not yet know their respective significance, but we can consider at least two possibilities: (i) The GPI specific T cells are able by themselves to damage or kill normal (GPI-positive) HSC, whereas the increased number of INFγ producing T cells is essentially an epiphenomenon; (ii) GPI specific T cells, after recognizing through their TCR the GPI presented by CD1d on normal (GPI-positive) HSC, are able to amplify their own cytotoxic action by recruiting a larger population of CD8+ T cells that is thus stimulated to produce INFγ. In conclusion, we have found that at least two distinct but functionally related auto-reactive populations of T cells are involved in the complex pathogenesis of PNH and IAA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.