Normal Hematopoietic Stem Cells Research Articles

Targeting HIF-2α for the Treatment of CML By Affecting LSCs Metabolism and the Vascular Microenvironment

Although the advent of tyrosine kinase inhibitors (TKI) drugs significantly increased the long-term survival of chronic myeloid leukemia (CML), residual LSCs can still lead to drug resistance and disease recurrence. As LSCs in CML rely on hypoxia-inducible factors (HIF) to maintain their own energy metabolism in the hypoxic environment of bone marrow, HIF-2α has not been systematically explored in CML. Here, we explored how HIF-2α participated in drug resistance and provided a potential new clinical strategy for CML. We first investigated the expression of HIF in the database and found the overexpression of HIF-2α in the bone marrow of CML patients compared with normal subjects, while there was no difference in HIF-1α and HIF-3α (Fig. 1A-B). We then collected bone marrow samples from normal subjects and CML patients. We found that compared with normal hematopoietic stem cells (Lin-CD90+CD34+CD38-), CML LSCs (Lin-CD90+CD34+CD38-) had higher levels of HIF-related proteins than common leukemia cells (Fig. 1C). To further explore the role of HIF-2α in the pathogenesis of CML, we constructed HIF-2α conditional knockout mice through transfecting the plasmid of Cre-BCR-ABL-GFP with transposase and Ctrl-BCR-ABL-GFP without transposition to HSPCs (Fig. 2A). After the first round of leukemia transplantation, the onset of CML was delayed in the HIF-2α knockout mice and the median survival time was prolonged from 31d in the Ctrl group to 60d (Fig. 2B). Next, we harvested fully onset CML mice for the secondary transplantation and found the median survival time extended from 46d to 117d in the HIF-2α knockout group (Fig. 2C). Then we investigate the biological function of HIF-2α in CML LSCs. We performed the clone formation assay on bone marrow cells of the first round of onset CML mice and found that HIF-2α knockout reduced the colony-forming ability of CML cells (Fig. 3A). To explore the tumorigenic ability of HIF-2α knockout, limiting dilution assay (LDA) was performed and found that the LSCs ratio of CML decreased by 30 times after HIF-2α knockout, from 1/76960 to 1/2237970 (Figure. 3B). Meanwhile, we explored the effect of HIF-2α knockout on the chemosensitivity in CML. TKI treatment combined with HIF-2α knockout can significantly improve the efficacy of TKI and extend the mouse survival time (Figure. 3C). To explore the specific mechanism by which HIF2A affects LSCs, we firstly investigate the effect of HIF-2α on LSCs metabolism. We detected metabolism-related indexes of LSCs in the CML model and found that the glycolysis ability of LSCs was weakened after HIF-2α knockout (Fig. 4A). At the same time, its oxidative phosphorylation pathway and reactive oxygen species (ROS) were enhanced (Fig. 4B-C) which may be an important cause of LSCs apoptosis. As HIF-2α can directly promote the transcription of pro-angiogenic factors, we detected the content of vascular endothelial growth factor (VEGF) α in the supernatant of mice bone marrow and found that it was significantly increased after the onset of CML but decreased after HIF-2α knockout (Fig. 5A). Fluorescent staining of bone marrow sections revealed that the density of blood vessels in bone marrow increased, whether in the intima (Fig. 5B) after the onset of CML. Next, we treated CML mice with VEGF Monoclonal antibody and found that it alone could prolong the survival time. However, the VEGF Monoclonal antibody did not affect HIF-2α knockout CML mice (Fig. 5C). These results suggest that HIF-2α is involved in bone marrow vascular remodelling after the onset of CML. To investigate the functional effect of HIF-2α in human CML, the CDX CML mouse model was made with CML cell lines (K562 and MEG-01) to observe the tumorigenesis of CML in nude mice, and they were treated with TKI. We found that HIF-2α knockout could delay the onset of CML and improve the therapeutic effect of TKI (Fig. 6A). To better apply the effect of HIF-2α on CML chemosensitivity to the clinic, we explored the effect of inhibitors of HIF, PT2399, on CML chemosensitivity. PT2399 can significantly increase the chemosensitivity of CML cell lines to TKIs and prolong the survival curve of CML mice treated with TKIs (Fig. 6B). In conclusion, HIF-2α knockout could inhibit the function of LSCs by affecting their metabolic pattern and vascular microenvironment in CML. Therefore, targeting HIF-2α may become a new strategy, and its specific inhibitor PT2399 may be a candidate drug for clinical application in CML treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Role of Stathmin-1 in the Regulation of Hematopoietic Stem Cells

Stathmin-1 is a cytoplasmic phosphoprotein that regulates microtubule dynamics via the promotion of microtubule catastrophe or the sequestration of alpha-beta tubulin heterodimers. Also known as Oncoprotein 18 (OP18), Stathmin-1 is highly expressed in a wide variety of tumors, including high-risk myelodysplastic syndromes and acute leukemias (Machado-Neto et al, 2014). Stathmin-1 is also highly expressed in normal hematopoietic stem cells (HSCs), and its loss is associated with impaired erythroid differentiation and altered megakaryopoiesis (Ramlogan-Steel et al, 2021). While these prior studies have established a role of Stathmin-1 in normal erythro-megakaryopoiesis, its role in hematopoiesis- particularly in hematopoietic stem and progenitor cells- is not clear. Given the high expression of Stathmin-1 in HSCs and the importance of microtubule dynamics to HSC differentiation, we hypothesized that Stathmin-1 loss may impact normal HSC function. To address the role of Stathmin-1 in HSCs, we characterized HSC phenotype and function in a Stathmin-1 knockout mouse (Stmn1-/-). Under homeostatic conditions, Stmn1-/- HSC number and frequency are normal compared to wild-type (WT) mice. However, Stmn1-/- HSCs have markedly impaired function, as their bone marrow (BM) failed to engraft in competitive transplants (10% vs. 60% chimerism at 24 weeks post-transplant p<0.0001). Competitive intra-tibia transplants were then done to circumvent potential homing defects, and again Stmn1-/- BM displayed a significant engraftment defect compared to WT (10% vs. 78% chimerism at 24 weeks p<0.0001). Of note, Stmn-1-/- HSCs performed equivalently to WT HSCs in an in vivo homing assay, further suggesting their lack of engraftment is not due to homing defects. Stmn1-/- BM cells were capable of engraftment in non-competitive transplants, albeit less robustly than WT cells (42% vs. 90% p<0.01). Treatment of WT and Stmn1-/- mice with one single dose of the chemotherapeutic agent 5-fluorouracil showed that Stmn1-/-mice have slower tri-lineage recovery than their WT littermates (p<0.01 at day 15). Consistent with their reduced in vivo function, Stmn1-/- HSCs produced fewer colonies than WT HSCs when plated in Methocult. They were also not capable of serial replating, supporting that loss of Stathmin-1 is associated with impaired HSC function. Consistent with its known role in regulating microtubules, Stmn1-/- HSCs displayed altered microtubule morphology by confocal microscopy with a lack of normal nuclear invaginations compared to WT HSCs. Analysis of cycling status revealed that Stmn1-/- HSCs are more quiescent than WT HSCs at baseline (1.3% vs. 3.4% at S/G2/M phase) and in response to 24 hours of G-CSF stimulation (3.4% vs. 7% at S/G2/M phase). In addition, Stmn1-/- HSCs have higher rates of apoptosis than their WT counterparts (18.5% vs. 9.90%), suggesting that Sthamin-1 loss leads to both impaired HSC cycling and increased cell death. We next performed RNA-seq on sorted Stmn1-/- and WT HSCs to gain further mechanistic insight into their impaired function. Differential pathway analysis showed alterations in genes associated with cellular metabolism, particularly mitochondrial function, and cell differentiation in the Stmn1-/- HSCs compared to WT controls. Consistent with these results, measurement of mitochondrial function (oxygen consumption rate) using the Seahorse analyzer showed that Stmn1-/- HSCs and progenitors have impaired maximal respiratory capacity compared to WT. Together, these data suggest a model whereby Stathmin-1 regulates microtubule dynamics in HSCs to promote optimal mitochondrial function to support cellular cycling, survival and self-renewal.

Stimulus MDS-US Trial in Progress: Evaluating Sabatolimab in Combination with Hypomethylating Agents (HMAs) in Patients with Intermediate-, High-, or Very High-Risk Myelodysplastic Syndrome (MDS)

Background Patients with intermediate-, high-, or very-high-risk myelodysplastic syndrome (I/H/vHR-MDS) need durable treatment options with favorable tolerability. Sabatolimab (MBG453) is a novel immunotherapy targeting TIM-3, an immuno-myeloid regulator expressed on immune and leukemic stem cells, but not on normal hematopoietic stem cells. Patients with higher-risk MDS experience poor outcomes and limited treatment options. HMAs are approved for the frontline management of higher-risk MDS, but 50% of HMA-treated patients experience primary failure, and most responders progress within 2 years. Novel therapies that provide improved durable outcomes with favorable safety profiles are needed for these patients. In a phase Ib study (NCT03066648), sabatolimab+HMAs demonstrated an encouraging safety-tolerability profile and emerging durable responses in patients with higher-risk MDS (64% overall response rate and approximately 84% of patients still in response after 6 months). Sabatolimab is being studied in multiple phase I-III trials within the STIMULUS program. We present the STIMULUS MDS-US trial, designed to further assess the safety and efficacy of sabatolimab+HMAs, including an oral HMA. Study Design and Methods: STIMULUS MDS-US (NCT04878432) is a nonrandomized, single-arm, open-label, phase II study evaluating the safety and efficacy of sabatolimab in combination with FDA-approved HMAs of the investigator's choice in patients with MDS in the United States. A 12-month extension phase will continue investigating safety and efficacy after 24 months of treatment. The planned enrollment is estimated to be 90 patients. The trial is currently enrolling. Eligible patients are aged ≥18 years and have treatment-naive, higher-risk MDS (IPSS-R intermediate- or high/very high-risk) not suitable for intensive chemotherapy or hematopoietic stem cell transplant. Patients will receive IV sabatolimab 800 mg on day 8 (D8; or once between D5-8 to coincide with partner HMA administration) of each 28-day cycle for 24 months plus the investigator's choice of the following HMAs: oral decitabine (Inqovi®, decitabine 35 mg and cedazuridine 100 mg, D1-5); IV decitabine (20 mg/m2, D1-5); or IV or subcutaneous azacitidine (75 mg/m2, D1-7 or D1-5+8-9). Primary end points are incidence and severity of adverse events (AEs) and serious AEs. Secondary endpoints are complete remission (CR) rate (according to International Working Group for the Prognosis for MDS), progression-free survival, overall survival, leukemia-free survival, percentage of patients with CR, marrow CR and/or partial remission, duration of CR, time to CR, and percentage of patients with improvement in red blood cell/platelet transfusion independence. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Three Gene Leukemic Stem Cell Signature Score Is Robustly Prognostic in Chronic Myelomonocytic Leukemia

Introduction Leukemic stem cells (LSCs) possess biological properties shared with normal hematopoietic stem cells. They confer resistance to chemotherapy and mediate relapse in acute myeloid leukaemia (AML) as well as in myelodysplastic syndromes (Ho et al., Blood 2016; Wang et al., Blood Advances 2020). A 17-gene stemness score ("LSC17") was predictive of treatment response and overall survival (OS) in AML (Ng et al., Nature 2016). Whilst chronic myelomonocytic leukaemia (CMML) is regarded as a myeloid stem cell neoplasm, the biological impact and prognostic implications of LSCs in CMML have not been explored. To this end, we evaluated the prognostic potential of these 17 genes in CMML patients. Methods We performed mRNA sequencing on bone marrow (BM) samples from 44 CMML patients at presentation, each with extensive clinical and laboratory correlates and longitudinal outcome data. CMML diagnosis was based on The International Consensus Classification (Arber et al., Blood 2022). The expression of each LSC17 gene was evaluated and integrated with clinical and outcome metadata. Results We identified three genes (MMRN1, ADGRG1, and DNMT3B) whose expression correlated significantly with overall survival (OS). An LSC-3 score was constructed based on the weighted sums derived from Cox regression analysis. Patients were divided into high and low LSC-3 score groups, with cut point determined by maximally selected rank statistics. Higher LSC-3 scores were associated with higher age (p=0.016), lower hemoglobin levels (p=0.011), and higher incidence of IDH1/2 mutations (p=0.009). High-score patients had significantly shorter leukaemia-free survival (LFS; median 7.4 months vs 30.5 months, p<0.001) and OS (10.5 months vs 33.9 months, p<0.001, Figure 1A). In subgroup analyses, LSC-3 score retained strong LFS and OS significance in both CMML-1 and CMML-2 stages as well as across dysplastic and proliferative CMML subgroups. Mutations in ASXL1 have been consistently associated with poor prognosis in CMML and incorporated into several existing prognostic systems (Patnaik, Haematologica2022). In patients with unmutated ASXL1, high LSC-3 score robustly correlated with inferior LFS and OS (p=0.001 and p=0.001, respectively). In multivariable analysis, higher LSC-3 score remained an independent adverse risk factor for LFS and OS, irrespective of CPSS and CPSS-Molecular risk status. Time-dependent ROC curve analysis suggested potential for LSC-3 score to improve current risk stratification systems, increasing area under curve when incorporated (Figure 1B). We validated the prognostic performance of the LSC-3 score in three independent CMML cohorts. Remarkably, LSC-3 score retained strong discriminatory performance even in a CMML monocyte RNA-seq dataset, suggesting potential application on peripheral blood samples. GSEA revealed enriched oxidative stress, altered metabolism, and aberrant immune and T-cell functions in LSC-3 high BM samples. CIBERSORTx analysis (Newman et al., Nature Biotechnology 2019) also indicated significantly lower fractions of activated NK-cells (p=0.026) and borderline significant fewer CD8 T-cells (p=0.06) in the BM from high-score CMML patients. Finally, single-sample GSEA highlighted differences in drug sensitivity profiles, including resistance signatures for hypomethylating agents and venetoclax in LSC-3-high patients, which might partially explain the dismal prognosis in this population. Discussion We describe a simple yet powerful three gene expression signature ("CMML-LSC3") as an independent and robust prognostic factor for LFS and OS in CMML patients. We validated this signature in three independent CMML cohorts with linked transcriptomic data, comprising both bone marrow mononuclear and sorted peripheral blood monocytes. We propose that LSC3 could be incorporated to complement existing scoring systems to improve prognostic performance and aid management decisions. Biologically LSC3 stratifies CMML patients by apparent differences in metabolism, immune microenvironment composition and drug resistance, raising the prospect of personalized therapeutic approaches mandating further study. The LSC3 score is being validated prospectively in a UK multicentre phase 2 trial (AMMO trial EudraCT 2021-004585-35). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Wnt and Notch Pathways Regulate Stemness of Myeloid Leukemia Cells Via IL33/ST2 Signaling

Therapy-resistant leukemic stem cells (LSCs) are a main cause of relapse in chronic and acute myeloid leukemia (CML and AML) patients. Stemness, a cellular state of LSCs characterized by their quiescence, pluripotency, and long-term self-renewal capacity, is thought to have major implications on therapy resistance and outcome in myeloid leukemias. However, signaling pathways that induce stemness are often not specific for LSCs and shared with normal hematopoietic stem cells (HSCs). Therefore, LSCs are difficult to be targeted therapeutically. IL-33/ST2 signaling may be a potential target to eradicate CML and AML LSCs. To investigate the ST2 gene (IL1RL1) expression in different hematological malignancies, we used the MILE gene expression dataset containing 2096 samples. The IL1RL1 high expressing samples (n=62) were exclusively represented by myeloid leukemias (CML and AML), and 50% of the IL1RL1 high samples harbored either the BCR/ABL1 (CML) or the AML1/ETO (AML) oncogenes. We confirmed the result from the MILE database by analyzing the ST2 protein expression on FACS-sorted CD34+ LSCs from AML and CML patient samples. Indeed, AML1/ETO and BCR/ABL1 harboring LSCs expressed ST2, while healthy hematopoietic stem cells (HSCs) and LSCs harboring other oncogenes were ST2-negative. Transfecting the ST2-negative cell line Ramos with the AML1/ETO and BCR/ABL1 oncogenes induced ST2-expression. Next, we performed a gene set enrichment analysis (GSEA), comparing the IL1RL1 high vs. IL1RL1 low samples in the MILE dataset. Genes involved in stemness (p<0.01), Wnt- (p<0.001), and NF-ΚB (p=0.054) signaling were upregulated, while the Notch-pathway (p<0.05) was downregulated. Since the NF-ΚB is a known downstream pathway of ST2, we hypothesized that Wnt and/or Notch signaling pathways regulate ST2 expression. Therefore, we treated FACS-sorted CD34+ AML1/ETO expressing LSCs with the Wnt pathway inhibitor XAV939 or HES1 and NOTCH1 shRNAs, silencing the Notch pathway. Treatment with XAV939 led to a decrease in ST2 expression in these cells, while silencing HES1 and NOTCH1 enhanced ST2-expression. To analyze the molecular mechanisms induced by IL-33/ST2 signaling, we performed a transcriptomic analysis using FACS-sorted CD34+ AML1/ETO LSCs upon treatment with or without IL-33. In total, 377 genes were up- and 139 genes downregulated after IL-33 stimulation. IL-33-induced genes were involved in the positive regulation of NF-ΚB (p<0.05) and canonical Wnt (p<0.001) signaling pathways, and the regulation of stem cell division and maintenance (p<0.001). To functionally test whether IL-33/ST2 signaling triggers stemness in myeloid leukemia, we performed a colony forming assay using CD34+ AML1/ETO or BCR/ABL1 expressing LSCs from leukemia patient samples. The colony numbers increased 1.5-3fold upon IL-33 treatment in three consecutive platings, indicating an increase in stem cell numbers. IL-33 had no effect on colony numbers of HSCs and LSCs harboring other oncogenes. To test the in vivo relevance of IL-33/ST2 signaling in leukemia development, we used a congenic AML1/ETO9a (AE9a) or BCR/ABL1 (CML) mouse model. Briefly, we transduced ST2-negative HSCs originating from BL/6 or ST2-knockout (ST2-/-) mice with AE9a-GFP or BCR/ABL1-GFP and transplanted those cells into recipients. In both models, the LSCs strongly expressed ST2, but IL-33/ST2 signaling had no influence on the disease development. Stemness supports resistance against standard therapy in both, AML and CML. Since we were able to show that IL-33/ST2 signaling promotes stemness in AML1/ETO and BCR/ABL1 LSCs, we hypothesized that IL-33 addition protects LSCs from treatment with standard therapeutics. Indeed, IL-33 rescued the reduced colony forming capacity of LSCs that were either treated with the chemotherapeutic agent cytarabine (AML1/ETO LSCs) or the tyrosine kinase inhibitor Nilotinib (BCR/ABL1 LSCs). Furthermore, ST2-deficiency led to a reduced LSC count in CML mice compared to control mice when treated with Nilotinib (p=0.047). We showed that ST2 is expressed on AML1/ETO and BCR/ABL1 harboring LSCs and that activated IL-33/ST2 signaling induced stemness in these LSCs. Targeting IL-33/ST2 signaling in these specific subtypes of AML and CML may eliminate LSCs and overcome resistance towards chemotherapy or TKIs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Anti-Myeloma Effect of Compound K and Rh2 in a Multiple Myeloma Xenograft Model

Introduction: Several ginsenosides have demonstrated anti-cancer activity due to their ability to inhibit DNA synthesis and angiopoiesis, inducing cancer death. Rh2, a PPD-type 20(S)-ginsenoside, has been extensively studied as a major ingredient with antineoplastic properties. Moreover, the characteristics of compound K (CK), a PPD ginsenoside metabolite, resulting from the metabolic activity of enterobacteria, have been investigated. The effect of ginsenoside has been actively studied in leukemia but not in multiple myeloma (MM). Thus, the aim of study was to identify ginsenosides that have toxic effects against MM cells and to elucidate the underlying molecular mechanism. Methods: First, we screened the toxic effects of ginsenosides Rb1, Rb3, Rg1, Rg3, Rh1, Rh2, and CK at a concentration of 50 μM in several MM cell lines (ARH77, U266, and RPMI8226) after 24 hours of exposure. Furthermore, ARH77 cells were incubated with Rh2 and CK at 0, 20 μM, 50 μM, 60 μM, and 70 μM for 24 hours, and the DNA content at each stage of the cell cycle was detected. The potential cytotoxic effect of Rh2 and CK was investigated in normal hematopoietic stem cells (HSCs) isolated from healthy donors using primary peripheral blood stem cells (PBSCs). For this experiment, HSCs were isolated using a PBSC collection procedure after the recruitment of healthy donors. Briefly, granulocyte colony-stimulating factor was administered continuously until the white blood cell count exceeded the threshold of 1 × 109 cells/L. The subjects underwent leukapheresis once or twice using a blood cell separator. To quantify the production of PBSCs from each leukapheresis, flow cytometry was carried out to quantify CD34+ cells. To establish a human MM xenograft model, RPMI8226-RFP-FLuc cells (5 × 106 per mouse) were intravenously injected into 9-12-week-old male and female NOD/SCID IL-2Rγnull (NSG) mice. To assess the anti-MM effect of CK and Rh2 in the MM xenograft model, NSG mice were divided into the following treatment groups: Experiment set 1: no treatment (PBS control), CK (15 mg/kg/day), Rh2 (15 mg/kg/day), K (30 mg/kg/day), and Rh2 (30 mg/kg/day). Mice were treated with either CK or Rh2 (15 or 30 mg/kg/day) by intraperitoneal injection for 3 days in a week at a 1-day interval. Experiment set 2: no treatment (PBS control), CK (7 mg/kg/day), Rh2 (7 mg/kg/day), and Lenalidomide (1 mg/kg/day). Mice were treated with either CK or Rh2 (7 mg/kg/day) by intravenous injection for 3 days in a week at a 1-day interval. Lenalidomide (1 mg/kg/day) treatment was given by oral administration from day 14 for 5 consecutive days in a week, and it was repeated for 3 weeks at a 2-day interval in each week. Tumor growth was monitored weekly by bioluminescence imaging in the dorsal view; 10 min before imaging, mice were intraperitoneally injected with D-Luciferin (150 mg/kg/mouse). Results: The ginsenosides Rh2 and CK inhibited the cell growth, exerted cytotoxicity, and induced G1 phase cell cycle arrest in human MM cells. However, these treatments demonstrated no cytotoxic effect on the self-renewal and the differentiation capacity of normal primary PBSCs. This observation implied that the cytotoxic activity of Rh2 and CK might selectively target MM cells and that these ginsenosides are safe in other cell types. Finally, we studied the anti-MM effect of CK and Rh2 in a clinically relevant human MM xenograft model. PBS was given in the no treatment control group and all untreated tumor-bearing mice showed rapid tumor growth and severe plasmacytoma, which led to death within 7 weeks. Notably, mice treated with CK and Rh2 showed significant inhibition of tumor growth and a longer survival. Conclusions: Ginsenosides CK and Rh2 inhibited the growth of MM cells in an in vitro and in vivo model without any significant cytotoxic effect on the self-renewal and differentiation capacity of PBSCs. Thus, these ginsenosides could be used in MM treatment and can be combined with drugs without exerting any severe toxic effects. Keywords: Ginsenosides; Rh2; Compound K; Multiple myeloma Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

In Search of Drivers of CD34+/CD38-/CD26+ Leukemia Stem Cells Persistence in CML Patients

Introduction: In Chronic Myeloid Leukemia (CML) patients, we confirmed that the expression of CD26 discriminates CML Leukemic Stem Cells (LSCs) from normal Hematopoietic Stem Cells (HSCs) or from LSCs of other myeloid neoplasms. CD34+CD38-CD26+ cells are easily measurable in Peripheral Blood (PB) at diagnosis, and circulating residual CD26+LSCs persist in most patients during treatment with Tyrosine Kinase Inhibitors (TKIs) and after successful TKI discontinuation. The reason for LCSs persistence may be correlated to mechanisms helping their survival, such as resistance to apoptosis (via BCL-2 or CD47 expression) or immune checkpoint escaping (via PD-L1). Moreover, few is still known about the "activity state" or "quiescence" of CML LSCs. Aim of the study: to unravel phenotypic characteristics that could help CML LSCs persistence, evaluating by flow-cytometry the expression level of BCL-2 protein, PD-L1, CD47 and Ki67 on CD26+ LSCs population. Methods: Flow-cytometry assays were performed on PB from newly diagnosed CML patients by using lyophilized reagents (CD34-FITC, CD26-PE, CD38-APC, CD45-V500) and liquid reagents (CD47-V450, PD-L1-Cy7, BCL-2-FITC, Ki67-PerCP-Cy 5.5). Analysis have been done using a BD FACSLyric cytometer. Results: 47 newly diagnosed CML patients entered this study. With regards to PD-L1 expression on CD26+LSCs, 22/47 (47%) scored negative while 25/47 (53%) resulted positive, albeit with a quite variable percentage of expression (median 28.5 % of CD26+ cells, range 12-82.8 %). BCL-2 protein was expressed in 47/47 (100%) CD26+ LSCs, and the level of expression evaluated comparing the Mean Fluorescent Index (MFI) was found much lower on CD26+ LSCs than lymphocytes (median 5.98, range 1.90-32.14; median 17.20, range 7-76, respectively, p value <0.05). Regarding CD47 expression, 47/47 (100%) patients scored positive, with an MFI that is double on lymphocytes compared to CD26+ LSCs (median 26.58, range 5.70-87.09; median 13.22, range 5.64-33.22, respectively). Lastly, all 47/47 (100%) CD26+ LSCs were found Ki-67 positive. Conclusions: These preliminary results show that in newly diagnosed CML patients, Ki67 is highly expressed on circulating CD26+ LSCs, suggesting an unexpected "active proliferative state" of CD26+ LSCs, rather than a "quiescent" status. The latter may explain the rapid reduction of the bulk of CD26+LSCs we observe already at 3 months of TKI treatment. Both BCL-2 protein and CD47 are expressed at a higher level on lymphocytes than on CD26+ CML LSCs, indicating that the "don't eat me signal" that blocks apoptosis may not be relevant for CML LSCs persistence. On the other hand, CD26+/PD-L1+ LSCs (found in about 53% of CML patients) could theoretically escape the immune checkpoint controls, an aspect that could be taken in consideration for a future Treatment Free Survival (TFR) approach in CML patients as it may lead to a loss of molecular response compared to patients with CD26+ LSCs scoring negative for PD-L1. Additional studies need to be performed on a larger cohort of patients with a longer follow-up to support these hypotheses; however, these preliminary data may start to pave the way to understand the relationship between CD26+ LSC and immunity of CML patients.

A Phase 1, Open-Label, Safety, Tolerability, and Efficacy Study of NC525 in Subjects with Advanced Myeloid Neoplasms

Background With the exception of certain select subtypes, Acute Myeloid Leukemia (AML) remains incurable for a majority of patients with current therapies. Despite recent drug approvals targeting actionable molecular mutations and BCL-2, relapse remains inevitable for most patients with AML due to leukemia-initiating cells and progenitors {i.e., leukemic stem cells (LSCs)} which are resistant to conventional cytotoxic drugs. Novel agents that preferentially target LSCs to lessen or eliminate the risk for relapse while avoiding significant myelosuppression by sparing normal hematopoietic stem cells (HSCs), are highly sought-after anti-leukemia therapeutics. Leukocyte Associated Immunoglobulin-like Receptor 1 (LAIR-1) is an immune inhibitory receptor that is present on most immune cell subsets, including T cells, NK cells, monocytes, macrophages, dendritic cells, and granulocytes. LAIR-1, a surface leukocyte receptor containing two immune receptor tyrosine-based inhibitory motifs (ITIM), binds to endogenous collagen ligands and plays an important role in inducing an immunosuppressive microenvironment. In AML, the engagement of LAIR-1 inhibits intracellular downstream survival signals, leading to decreased GM-CSF-mediated survival and proliferation of AML blasts. LAIR-1 is expressed on AML blasts and LSCs regardless of FAB subtypes, cytogenetics or molecular mutations. Recent work demonstrates that myeloid leukemia cell lines, primary AML blasts, monoblasts and monocytes, and MDS-EB-2 blasts all express LAIR-1. Critically, while high expression of LAIR-1 is characteristic of LSCs, this receptor is minimally detectable on normal HSCs, making it an ideal anti-leukemic target. NC525 is a humanized immunoglobulin gamma 1, kappa (IgG1κ) monoclonal antibody that targets LAIR-1-expressing AML blasts and LSCs. Extensive IND-enabling preclinical studies have shown the following features of NC525: (i) Inhibition of colony formation of AML CD34+ bone marrow cells; (ii) sparing of colony formation and differentiation ability of healthy CD34+ HSCs; (iii) direct ex vivo killing of AML blasts without killing healthy leukocytes; (iv) suppression of AML in both MV4-11 and THP-1 CDX models; (v) restriction of MV4-11 in a humanized murine model, without depletion or suppression of human T cells; (vi) restriction of MV4-11 tumor growth in murine blood, spleen, and bone marrow; and (vii) reduction of AML disease burden in primary patient-derived AML xenograft (PDX) models. Finally, NC525 completely inhibits leukemia propagation in secondary transplant models, suggesting that NC525 is capable of eradicating LSCs. Furthermore, NC525 destroys leukemic cells by ligation-mediated apoptosis of leukemic blasts and stem cells without evidence of an immunomodulatory effect on other immune subsets. Methods This is an open-label, non-randomized, Phase 1 dose escalation study to determine the safety and tolerability of NC525 in adult patients with advanced myeloid neoplasms. This study will also assess the clinical benefit of NC525. Eligible patients must have relapsed or refractory AML, myelodysplastic syndrome (MDS), or chronic myelomonocytic leukemia (CMML) that has progressed after standard therapy. Participants will receive NC525 by intravenous infusion on Days 1 and 15 of each 28-day cycle. During Phase 1, the safety and tolerability of NC525 will be evaluated by exploring escalating dose levels of NC525. In addition, the pharmacologically active dose (PAD), defined as the dose that provides a maximal biologic effect, such as achieving a complete remission (CR), CR with partial hematological recovery (CRh) or CR with incomplete hematological recovery (CRi), and the maximum tolerated dose (MTD) of NC525 will be determined. Safety and tolerability will be further assessed through the expansion of a few dose levels to determine an optimal recommended Phase 2 Dose (RP2D) and administration schedule of NC525, based on the PK/PD profiles. Taken together, preclinical data suggest that targeting LAIR-1 in AML and other myeloid malignancies may be an effective therapy for these diseases with the advantage of specifically eradicating leukemic stem cells and sparing normal HSCs.

EVI1 Promotes Immune-Evasive Microenvironment Via Cyclin D1 in Acute Myeloid Leukemia

Inappropriate expression of Ecotropic viral integration site 1 (EVI1) has been associated with dismal clinical outcomes in acute myeloid leukemia (AML), while EVI1high (EVI1+) AML cells do not show clear resistance to cytotoxic agents in vitro. Since EVI1 is essential for normal hematopoietic stem cells (HSCs), we performed chromatin-immunoprecipitation (ChIP)- and RNA-sequencing of murine EVI1+ AML cells and normal hematopoietic cells to identify factors involved explicitly in AML, and thereby to elucidate molecular cues to refractory EVI1+ AML. We firstly employed a murine EVI1-transformed AML (EVI1-AML) model, where murine hematopoietic cells exogenously expressing 3×FLAG-tagged EVI1 were transplanted into syngeneic mice. Using EVI1-AML cells, we next performed ChIP coupled to next-generation sequencing (ChIP-seq) by anti-FLAG tag antibody and transcriptome analysis by RNA-seq. We further generated another murine EVI1+ AML model by retrovirally transducing the KMT2A-MLLT1 fusion gene into Lineage- Sca-1+ c-kit+ cells from EVI1-GFP knock-in mice, where Evi1-IRES-GFP allele was inserted into the Evi1 locus to enable real-time monitoring of Evi1 expression by GFP, followed by transplantation into lethally irradiated syngeneic mice. Intriguingly, GFPhigh L-GMPs from KMT2A-MLLT1 AML mice showed distinct expression profiles from GFPlow L-GMPs, including high NF-kB activity and low stem cell activity, along with the lower frequency of leukemia-initiating cells in GFPhigh fraction. Consistently, an exogenous expression of EVI1 in KMT2A-MLLT1 AML cells without Evi1 expression decreased colony-forming and leukemia-initiating activity, suggesting a differential role of EVI1 in AML from normal HSCs, where EVI1 is essential in maintaining stem cell function. Combined with functional screening by cell proliferation-related short hairpin RNAs (shRNAs), we identified that cyclin D1 (CCND1) was a downstream target, commonly implied in the above analyses, and a potential therapeutic vulnerability of EVI1-AML. EVI1 bound to the region 3.8 kb upstream of the transcription start site of Ccnd1, through which EVI1 demonstrated strong transcriptional activity in a luciferase reporter assay. Genetic silencing of Ccnd1 and pharmacologic inhibition of Cyclin D1 in EVI1- and EVI1+ KMT2A-MLLT1 AML cells negatively affected proliferation and colony-forming activity in vitro. Moreover, in vivo knockdown of Ccnd1 strikingly impaired the leukemogenic capacity of murine EVI1-AML models. It was surprising that transcriptomic analysis of EVI1-AML cells with Ccnd1 silencing showed the downregulation of genes involved in chemokine production and response to interferon instead of cell cycle-related genes. The expression of Cd274 (PD-L1) as well as Stat1, the primary signaling mediator for type I and II IFN, was decreased in EVI1-AML cells with Ccnd1 knockdown. EVI1 directly bound to the promoter region of Stat1 and activated its transcription in line with a previous report, and indirectly up-regulated STAT1 expression via Cyclin D1. Accordingly, infiltrating CD4 and CD8 T cells from EVI1-shCcnd1 AML mice demonstrated decreased exhaustion markers, including LAG3, PDCD1, and TIGIT. Silencing of Stat1 and Ifngr, the receptor for IFN-γ, impaired the leukemogenic capacity of EVI1-AML in vivo, with a decrease in T and NK cells with exhaustion phenotypes, while Ifnar, the receptor for IFN-α, was not involved in this process. Genetic inhibition of the IFN-γ-STAT1 axis in non-EVI1+ AML models did not affect AML development, nor the frequency of immune cells expressing exhaustion-related molecules, suggesting this axis is specifically important in EVI1+ AML. By using human AML datasets, overexpression of EVI1 and CCND1 were associated with IFN-γ signature, including up-regulation of STAT1 and CD274, and elevated expression of chemokines, with increased exhaustion molecules in T cells. These data collectively suggest that cyclin D1 is implicated in the development of EVI1-AML through the formation of IFN-γ signature and exhausted T cell phenotypes, which could be potentially targeted.

Elongation of Long-Chain Fatty Acids Is Crucial for Hematopoietic Stem Cell Engraftment and Leukemia Propagation

Lipid metabolism plays a crucial role in regulating physiological stem cells and cancer cells. Variations in length or degree of desaturation of fatty acid (FA) chains greatly contribute to lipid diversity, but whether it significantly regulates hematopoietic cell biology remains unknown. The enzyme ELOVL6 converts C16 saturated and monounsaturated FAs to C18 species. Elovl6 gene disruption (Elo6KO) in mice decreases the proportions of stearate (C18:0) and oleate (C18:1n-9) and increases those of palmitate (C16:0) and palmitoleate (C16:1n-7). Here, we report that ELOVL6 is essential for hematopoietic stem cells (HSC) engraftment after bone marrow (BM) transplantation and acute myeloid leukemia (AML) development in a mouse model. To determine whether hematopoietic cells of Elo6KO mice exhibit changes in FAs composition, we analyzed FAs in BM cell lysates. We found that the ratio of stearate to palmitate (C18:0/C16:0 ratio) was significantly decreased in Elo6KO cells compared to WT cells. To assess the effects of Elovl6 loss on the mouse HSC, we performed competitive repopulation assays. Using control or Elo6KO donor BM cells mixed in a 1:1 ratio with competitor BM cells, the chimerism of peripheral blood (PB) was about 50% in mice transplanted with control cells but remained at < 0.2% in those with Elo6KO cells at 12 weeks after transplantation. Even when transplanted Elo6KO donor cells with the competitor in a 10:1 ratio, the chimerism of PB was similarly low. Next, we investigated the roles of Elovl6 in leukemogenesis. We infected lineage-negative and c-Kit+ cells from WT or Elo6KO BM with MLL-AF9(MA9) retrovirus and transplanted lethally irradiated mice with either WT or Elo6KO MA9 cells. All mice receiving WT MA9 cells developed AML; however, AML propagation in Elo6KO MA9 cell-transplanted recipients was blocked. Elo6KO MA9 cell-transplanted mice survived until 6 months after transplantation. To assess the mechanisms underlying these phenotypes, we performed RNA-seq and gene enrichment analysis (GSEA) using CD34-, lineage-negative, Sca-1+ and c-Kit+ (CD34-LSK) cells and MA9 cells from Elo6KO or control mice. Chemotaxis, CXCR4 pathways, and their related target gene sets were enriched in Elo6KO cells, relative to control in both CD34-LSK cells and MA9 cells. We tested the chemotaxis of Elo6KO CD34-LSK and MA9 cells toward CXCL12 in a migration assay. A greater fraction of WT CD34-LSK and MA9 cells than that of these Elo6KO cells migrated to the CXCL12-containing chamber. We then examined the pharmacologic effects of inhibiting the PI3K pathway on chemotaxis. In a migration assay, the presence of PI3K inhibitor, Copanlisib, decreased the migration rate in WT MA9 cells, but not significantly in Elo6KO MA9 cells, which was already compromised. By contrast, AKT inhibitor, MK2206, did not alter WT or Elo6KO MA9 cell migration. In GSEA, Rac1-related pathways were enriched in Elo6KO cells. We thus investigated whether Rac1 activation was blocked by Elovl6 loss. The levels of the GTP-bound Rac1 were markedly increased in WT MA9 cells following CXCL12 stimulation compared to control, while those were not significantly different with/wo CXCL12 stimulation in Elo6KO MA9 cells. Given impaired chemotaxis and CXCR4-PI3K-Rac1 signaling in Elo6KO cells, we then investigated whether these cells exhibited altered actin remodeling in response to CXCL12. As expected, CXCL12 treatment induced lamellipodia formation in WT CD34-LSK cells, but not in Elo6KO CD34-LSK cells. Thus, ELOVL6 may regulate a CXCL12-CXCR4 pathway, resulting in PI3K-Rac1-mediated cytoskeletal remodeling and chemotaxis. Finally, to determine whether exogenously expressed Elovl6 could rescue Elo6KO MA9 cell phenotypes, we generated Elo6KO MA9 cells expressing Flag-tagged ELOVL6 (Elo6KO MA9Elo6) and mock controls (Elo6KO MA9mock). To evaluate the rescue of AML propagation, we transplanted lethally irradiated syngeneic mice with either Elo6KO MA9mock or Elo6KO MA9Elo6 cells. Among Elo6KO MA9mock transplanted mice, few recipients developed AML, while 10 out of 14 recipients had died due to AML after Elo6KO MA9Elo6 cell transplant. In summary, this study demonstrates novel and unexpected outcomes in normal HSCs and AML driver-transformed cells following lipid changes induced by Elovl6 deficiency. Altering lipid diversity could be a potential strategy to treat AML.

Single Cell Analysis Identifies Immune Marker IL4I1 As Critical for Leukemogenesis and Immunity

The treatment landscape of acute myeloid leukemia (AML) is evolving with new targets entering clinical translation. However, patients’ responses are still heterogeneous. Bone marrow (BM) is considered an immune privilege organ, but recent evidence suggests that inflammatory insults can increase cell cycling in hematopoietic stem cells (HSCs) gradually accumulating DNA damage. Thus, discovering immune targets on leukemia cells is an urgent unmet need. Here, we used scRNA seq to compare BM aspirates from 12 complete response (CR) and 10 refractory patients. We found 24 cell clusters expressing established markers of hematopoietic population. Differentially expressed genes between CR and refractory patients were determined using EdgeR and illustrated by volcano plots (Log2FC>|0.58|, x-axis, and Log10Adjusted P, y-axis) followed by enrichment for immune markers. Interleukin 4-induced gene 1 (IL4I1), in cluster 5 (promonocyte-like AML) was the most significant immune marker overexpressed in refractory patients (Fig. 1A). IL4I1 encodes a secreted oxidase protein (Molinier-Frenkel et al. FEBS 2017) that induces cancer immune escape by limiting proliferation of cytotoxic T cells (Lasoudris et al. Eur J Immunol. 2011). IL4I1 also activates the aryl hydrocarbon receptor and correlates with lower survival in glioma patients (Sadik et al. Cell. 2020). Its role in AML is unknown. To determine its clinical relevance in AML, we generated Kaplan-Meier curves using TCGA (n=132) and TARGET AML (n=187) databases and showed that patients with high IL4I1 had decreased survival (Fig. 1B&C). IL4I1 expression is increased in AML patients’ samples as compared to normal blood samples (Fig. 1D) and found in patients with mixed lineage leukemia (MLL) or complex cytogenetics (Fig. 1E). We next interrogated IL4I1 expression on leukemia stem cells (LSCs) defined as CD34+CD38- in the Princess Margaret Leukemia database (n=75) and noted IL4I1 expression was higher on LSCs vs normal HSCs (Fig. 1F). We then knocked out the IL4I1 gene in MOLM14 leukemic cells using CRISPR/Cas9 technology. IL4I1 silencing inhibited leukemia proliferation measured by Ki67 (Fig. 1G) and increased leukemia apoptosis measured by Annexin-V (Fig. 1H) compared to wild type (WT) leukemic cells. IL4I1 deficiency in leukemic cells arrested G2/S/M cell cycle progression which accelerated cell differentiation as compared to WT leukemic cells (Fig. 1I). We also performed Pearson correlations of IL4I1 with leukemic oncogenic drivers such as homeobox A cluster (HOXA) genes which strongly associate with the MLL translocation (Ernst et al. Curr. Biol. 2004) in TCGA- and TARGET-AML databases. Radar plots showed that IL4I1 was significantly correlated with these HOXA family genes (Fig. 1J, TCGA shown). We next interrogated immune regulatory functions of IL4I1 in AML patients and showed that enrichment of IL4I1 high genes vs low were in 4 signaling pathways: (i) cytokines/inflammation, (ii) innate immunity, (iii) antigen presentation and (iv) adaptive immunity (Fig. 1K). Representative genes signatures in these 4 pathways are summarized in Fig. 1L. We further tested whether IL4I1 expressing leukemia cells (WT) suppress T cells expansion in vitro. We used CFSE dilution to measure CD8+ T cells proliferation. WT MOLM14egfp cells inhibited CD8+ T cell proliferation while IL4I1 blockade in MOLM14egfp cells restored their proliferation (Fig. 1M). To test the role of IL4I1 in AML progression in vivo, we injected NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice with IL4I1-CRISPR/Cas9 KO or WT MOLM14egfp cells (Fig. 2A). Mice injected with WT MOLM14egfp cells had higher leukemia burden (Fig. 2B), higher Ki67 proliferation rate (Fig. 2C), and lower apoptosis rate (Fig. 2D) than IL4I1-KO MOLM14egfp recipients at Day 14 post-challenge. Self-renewal ability of leukemia cells is correlated to reactive oxygen species (ROS) (Testa U. et al. Exp Hematol. 2016). We found that ROS levels in IL4I1 KO leukemic cells are diminished compared to WT leukemic cells (Fig. 2E). Finally, we tested IL4I1 expression in response to Fedratinib, a JAK2 inhibitor, and showed that IL4I1 expression was decreased in responders (R) vs nonresponders (NR) (Fig. 2F). We conclude that IL4I1 is a rational immune therapeutic target for AML and testing of IL4I1 Inhibitor CB-668 through a collaboration with Calithera is underway. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Modeling Premalignant Transformation of Hematopoietic Stem Cells in a Nanobioreactor in Microgravity

Microgravity coupled with increased radiation exposure aboard the ISS provides a unique environment to simulate and study response to injury, inflammatory signaling, aging, and (pre-)malignant transformation of normal human hematopoietic stem cells (HSCs) in an accelerated timeframe. The NASA Twin study suggests that genomic, epigenomic, epitranscriptomic, and proteomic changes may detrimentally impact hematopoietic stem and immune cell fitness and induce stem cell exhaustion (Garrett-Bakelman et al., Science, 2019). Moreover, changes indicative of pre-cancer stem cell generation, such as increased chromosome translocations and inversions, occurred and persisted post-flight. Additionally, a recent publication entitled Multisystem Toxicity in Cancer: Lessons from NASA's Countermeasures Program found significant similarities between the multisystem physiological toxicities experienced in cancer patients as well as during spaceflight (Scott et al, Cell 2019). These reports highlight the benefits of studying injury response, changes in mutational profiles and cancer evolution in microgravity at the stem cell level. For this study, we designed a novel bioreactor system to support the culture of donor-derived human HSCs in low Earth orbit (LEO). A sponge matrix and stromal cells model the microenvironment HSCs reside in within the bone marrow niche. Testing on Earth confirmed our system's ability to maintain stem cell fitness over several weeks. To assess stem cell physiology in LEO over time, we lentivirally transduced a reporter into the HSCs pre-flight (Pineda et al., Scientific Reports 2016), which allows for cell cycle tracking via fluorescence imaging. This will provide data for assessment of stem cell health, maintenance and functionality. Furthermore, we are analyzing cytokine profiles and mutational status post-flight, with a focus on signatures we have previously connected to (pre-)malignant transformation via RNA sequencing analysis (Jiang, Cancer Cell 2019). The first batch of bioreactors launched with the SpX-24 mission in December 2021 and the second batch is currently on orbit as part of the SpX-25 mission. This investigation has the potential to provide valuable insights into the maintenance of hematopoietic stem cell health and functionality, HSC response to injury through accumulation of mutations and, eventually, the mechanisms fueling long-term (pre-)malignant transformation into leukemia stem cells.

Identification of Global miRNA Targetomes within Human Hematopoietic Progenitors and Acute Myeloid Leukemia Uncovers Unique Molecular Networks Centered on Mitochondria Function in Leukemic Stem Cells

Previous studies comparing transcriptome and proteome profiles of the cells that constitute both the normal and malignant hematopoietic hierarchies have revealed substantial uncoupling of transcript and protein expression levels. Despite the widely established role of miRNAs in regulating many cellular functions, the precise contribution of miRNA-mediated post-transcriptional regulation linking gene expression and proteome signatures of hematopoietic stem cells (HSC) and leukemic stem cells (LSC) remains largely unknown. Previous efforts to characterize miRNA targets have largely relied on computational prediction algorithms, which suffer from high rates of false-positive and negative target predictions and ultimately preclude discovery of non-canonical miRNA-target interactions. In this study, we perform Argonaute 2 (AGO2) crosslinking and immunoprecipitation (CLIP)-seq to experimentally map endogenous miRNA-target interactions in human CD34+ hematopoietic stem and progenitor cells (HSPC) from cord blood and six primary AML patient samples in an unbiased, transcriptome-wide manner (Manakov et al. 2022). Using this approach, we identify hundreds of miRNAs directly ligated to thousands of unique targets from miRNA-target chimeric sequencing reads. We find limited overlap (~30%) between miRNA targets captured by CLIP-seq and computationally predicted targets, underscoring the importance of functional validation of miRNA targets in specific cell types. Surprisingly, we find remarkable similarity between abundant miRNAs from HSPC and AML samples, including miR-142 and miRNAs encoded by the miR-17-92 cluster. However, the targets and the gene regulatory networks controlled by these highly abundant miRNAs are vastly different between HSPC and AML. In HSPC, we find that targets of abundant miRNAs are enriched in cell cycle regulation, histone modification and megakaryocyte/myeloid differentiation. In contrast, targets of the same miRNAs are enriched in protein localization/translation, neutrophil function, regulation of apoptosis and electron transport chain (ETC) pathways in AML. Collectively, we find that the composition of the abundant miRNAs is similar in normal HSPC and AML tumors; however they control divergent gene regulatory networks. To identify miRNA-target networks that play functional roles in LSC, we performed small RNA sequencing in 23 AML patient samples where each sorted fraction was assessed for LSC content by xenotransplantation. We derived an LSC-specific miRNA signature consisting of 5 miRNAs (LSC miR-5 score) predictive of overall survival and capable of stratifying AML patients independent of mRNA expression and other established indicators of outcome. Comparison of CLIP-seq targets of the LSC-specific miRNAs from HSPC and AML patient blasts revealed the existence of AML-specific gene regulatory networks centred on oxidative phosphorylation, ETC and cell-matrix adhesion. We further interrogated the functional significance of one miRNA from the LSC miR-5 score, miR-let-7c. We find that miR-let-7c is highly expressed in LSC-enriched populations of two AML in vitro models that recapitulate the leukemic hierarchy. Enforced expression of miR-let-7c in OCI-AML22 cells resulted in expansion of the LSC-enriched CD34+CD38- population and reduction in the CD34- terminally differentiated blast population. Comparison of miR-let-7c targets from HSPC and AML patient samples revealed the existence of distinct gene regulatory networks. In HSPC, miR-let-7c targets are enriched in cell cycle and regulation of transcription, whereas in AML, mitochondrial ETC, oxidative phosphorylation, and cell-matrix adhesion were the most enriched pathways. Moreover, analysis of miR-let-7c targets identified a high prevalence of transcripts encoded by the mitochondrial (mt) DNA (MT-ATP6, MT-ATP8, MT-ND1, -2, -4, -5, MT-CO1), pointing towards a largely uncharacterized function of miRNAs in regulating transcripts encoded by mtDNA in AML. Collectively, miRNA sequencing in LSC-enriched AML samples and identification of miRNA targetomes in HSPC and AML patient samples have revealed unexpected mechanisms of post-transcriptional regulation in normal and malignant hematopoietic stem cells.

Thrombopoietin Receptor Signaling Promotes the Competitive Advantage of TET2-Mutated Hematopoietic Stem and Progenitor Cells in Clonal Hematopoiesis

Clonal Hematopoiesis of Indeterminate Potential (CHIP) is an age-related condition that predisposes individuals to blood cancers. The time interval from acquisition of CHIP to overt neoplasia can span many years, providing an opportunity to delay or prevent malignant transformation. A key to achieving this goal is identifying differential dependencies between mutant and normal hematopoietic stem and progenitor cells (HSPCs). Targeting such dependencies may prevent against CHIP-related health risks. One of the most commonly mutated genes in CHIP is TET2, which encodes a methylcytosine dioxygenase. In mouse models, Tet2 knockout (KO) HSPCs have a competitive advantage over wild-type (WT) HSPCs. In this study, we aimed to identify differential dependencies between Tet2KO and Tet2WT HSPCs. We first generated isogenic murine cell lines by overexpressing HOXB4, a transcription factor involved in self-renewal, in HSPCs derived from a Tet2KO mouse and Tet2WT littermate. Using the isogenic cell lines termed HPCHOXB4, we conducted an epigenetic drug screen and found SGC0946, a selective inhibitor of H3K79 methyltransferase DOT1L, reduced the advantage of Tet2KO HPCHOXB4 cells in competition assays by preferentially reducing their proliferation and viability. SGC0946 also reduced the competitive advantage of unmodified Tet2KO HSPCs in competition assays, indicating that the dependency was not a consequence of HOXB4 overexpression. To decipher the mechanism behind this differential dependency, we performed RNA-seq and ChIP-seq for H3K79me2 on Tet2KO and Tet2WT HPCHOXB4 cells at baseline and after treatment with SGC0946. Expression of Mpl, which encodes the thrombopoietin receptor TPOR, was 20-fold higher in Tet2KO HPCHOXB4 than in Tet2WT cells at baseline and decreased after drug treatment. Mpl expression at the mRNA and protein level was also elevated in unmodified Tet2KO HSPCs compared with Tet2WT cells. Importantly, ChIP-seq analysis revealed that the level of H3K79me2 which associates with active transcription at the Mpl gene locus was higher in Tet2KO than in Tet2WT HPCHOXB4 cells at baseline, a difference that was erased with SGC0946 treatment. Based on the above findings, we hypothesized that Tet2KO HSPCs are more dependent on TPOR signaling than Tet2WT cells. Consistent with our hypothesis, a higher concentration of TPO in the culture medium increased the competitive advantage of Tet2KO HPCHOXB4 cells. Moreover, overexpression of Mpl in Tet2KO HPCHOXB4 cells rescued the effects of DOT1L inhibition, and shRNA-mediated suppression of Mpl expression was sufficient to decrease the competitive advantage of Tet2KO HPCHOXB4 cells and unmodified Tet2KO HSPCs over their corresponding Tet2WT counterparts. To explore the translational relevance of our findings, we tested if inhibition of JAK2, a critical mediator of TPOR intracellular signaling, would also reduce the competitive advantage of Tet2KO HSPCs. In line with our hypothesis, treatment with JAK2 inhibitors (fedratinib and AZ960), but not JAK1 inhibitors, suppressed the competitive advantage of Tet2KO HPCHOXB4 cells in vitro. Importantly, competitive repopulation studies showed that treatment with ruxolitinib, a clinically approved JAK1/2 inhibitor, also reduced the competitive advantage of untransformed Tet2KO HSPCs in vivo. To determine if our findings apply to humans, we generated two TET2KO cell pools from cord blood HSPCs using CRISPR/Cas9. The TET2KO cells expressed higher levels of TPOR and were more sensitive to treatment with SGC0946 and ruxolitinib compared with control cells. In addition, we expanded HSPCs from TET2-mutated mobilized peripheral blood samples in short-term cultures and monitored the size of the mutant clones by measuring the allele frequency of the sample-specific TET2 variants. In untreated cultures, the relative size of the mutant clones increased over time, an effect that was diminished by treatment with either SGC0946 or ruxolitinib. In summary, our findings demonstrate that TET2-mutated HSPCs are more dependent on TPOR signaling than TET2WT HSPCs. This differential dependency is mediated through increased H3K79 methylation at the MPL gene locus and enhanced expression of MPL. Furthermore, inhibition of the TPOR/JAK2 signaling axis is effective in suppressing the competitive advantage of TET2-mutated HSPCs, thus identifying a tractable therapeutic target for TET2-mutated CHIP.

PDZD2 Is Essential for Steady-State Hematopoiesis and Its 37-Kda Secreted Product, sPDZD2, Functions As a Soluble Tumor Suppressor in AML

PDZ domain-containing protein 2 (PDZD2) is expressed in normal hematopoietic stem and progenitor cells (HSPC), specifically HSC, MPP and erythroid progenitors, however, it is almost universally lost through hypermethylation in myeloid malignancies. PDZD2 encodes for a 300kDa protein containing 6 PDZ domains. The full-length protein is cleaved in the cytoplasm in a caspase 3-dependent manner, and the C-terminal fragment containing PDZ domains 5 and 6 is secreted to the tissue microenvironment as a 37kDa protein known as secreted PDZD2 (sPDZD2). Given that sPDZD2 has been reported to function as a tumor suppressor in solid tumors and its almost universal loss in AML, we hypothesized that PDZD2 is required for normal hematopoiesis and that sPDZD2 functions as a soluble tumor suppressor in the hematopoietic system. To determine if sPDZD2 is secreted to the bone marrow (BM) microenvironment we analyzed sPDZD2 levels in BM plasma isolated from primary AMLs and healthy donors using an ELISA-based detection method. We confirmed sPDZD2 secretion by normal CD34+ HSPC and detected significantly lower levels in AMLs (169 ± 17.53 vs 46.12 ± 6.70 ng/ml; p<0.0001). In addition, AML cell lines secreted significantly lower levels of sPDZD2 into the culture medium than HSPCs (28.71 ± 1.76 vs 4.62 ± 1.19 ng/mL; p<0.0001). Notably, treatment of a panel of AML cell lines (n=9) and primary human AML cells (n=4) with recombinant sPDZD2 (r-sPDZD2; dose: 100nM-300nM/day) led to growth inhibition (CellTiter-Glo, p<0.05 for all), differentiation (confirmed by upregulation of CD15/CD11b by flow cytometry) and cell cycle arrest in G0/G1. qRT-PCR analysis of cell cycle regulators in MV4-11 cells treated with r-sPDZD2 confirmed upregulation of CDKN2A (4.01-fold) and E2F4 (4.66-fold) and marked downregulation of multiple cyclin genes, including cyclins D and E, compatible with the cell cycle arrest observed by flow cytometry. To identify signaling pathways downstream of sPDZD2, we treated MV4-11 cells with 100nM r-sPDZD2 and performed phospho-kinase protein array followed by immunoblot for validation. In vitro treatment with r-sPDZD2 led to inhibition of ERK1/2 (T202/Y204, T185/Y187) and CREB (S133) phosphorylation, as well as decreased ß-Catenin expression and increased GSK3ß (S9), RSK1/2 (S221/S227) and p53 (S46) phosphorylation. In order to determine whether PDZD2 plays a role in normal hematopoiesis we performed in vitro differentiation in CRISPR-edited human HSPCs. We used a Cy3-labeled recombinant Cas9 protein and specific single guide RNAs (gRNAs) targeted against PDZD2. Cy3-positive cells were then used for in vitro myeloid or erythroid differentiation, and cell surface markers were evaluated by flow-cytometry analysis over a period of 11 days. While PDZD2-edited cells (PDZD2 KO) displayed a minor defect on myeloid differentiation due to insufficient upregulation of CD15, erythropoiesis appears to be more compromised, with a significant failure to adequately upregulate both CD235a (39 vs 15%) and CD71 (58 vs 26%). To determine the role of Pdzd2 in vivo, we took advantage of a Pdzd2Gt/Gt gene-trap knock-out mouse model and performed peripheral blood (PB) and BM analysis. PB counts at 8-weeks of age showed anemia, with a significant decrease in both red blood cell counts (9.04 ± 0.26 vs 7.58 ± 0.47 x106/µL) and hemoglobin levels (14.88 ± 0.46 vs 12.30 ± 0.81 g/dL) (p<0.05 for both), an observation compatible with the impaired erythropoiesis seen in vitro after PDZD2 KO in human HSPCs. In addition, we observed a trend to decreased MEPs and short-term HSCs, in the BM of 12-week-old mice (n=3), though the small size of the cohort prevented robust statistical analysis. In summary, our findings are the first to shed light on a previously unrecognized role for PDZD2 in hematopoiesis. Loss of PDZD2 in HSPC resulted in impaired erythroid differentiation both in vitro and in vivo. Moreover, our findings validate a soluble tumor suppressor role for sPDZD2 in AML, similar to that seen in solid tumors. Importantly, we further demonstrate a potential role for recombinant sPDZD2 as a novel therapeutic approach for AML.

Simultaneous Inhibition of SIRT3 and Cholesterol Homeostasis Targets AML Stem Cells By Perturbing Fatty Acid β-Oxidation and Inducing Lipotoxicity

Outcomes for patients with acute myeloid leukemia (AML) remain poor due to the inability of current therapies to fully eradicate disease initiating leukemia stem cells (LSCs) (Shlush et al., 2017). Oxidative phosphorylation (OXPHOS) is a highly targetable and essential process for LSC energy production and survival (Lagadinou et al., 2013). Sirtuin 3 (SIRT3), a mitochondrial deacetylase with a multi-faceted role in metabolic regulation (Finley et al., 2016), has been specifically shown to regulate OXPHOS in cancer models (Li et al., 2019, Xu et al., 2019). Thus, we sought to identify if SIRT3 is essential for LSC function. To determine if SIRT3 is essential for LSCs, we measured cell viability, colony forming potential, and engraftment potential of primary AML specimens upon perturbation of SIRT3 function using both RNAi and a small molecule inhibitor (YC8-02) (Li et al., 2019). SIRT3 reduction significantly decreased viability, colony forming potential and engraftment of LSCs (Fig. 1A), suggesting that SIRT3 inhibition decreases LSC function. Importantly, neither SIRT3 knockdown nor inhibition impacted the viability, colony forming potential, or engraftment potential of normal hematopoietic stem and progenitor cells (HSPCs). Altogether, these data suggest that SIRT3 is essential for LSCs and not HSPCs. To determine the molecular mechanisms by which SIRT3 is essential for LSC function, we performed proximity-dependent biotin labeling (BioID) in 293 T-Rex cells to map the interactome of SIRT3. Strikingly, our BioID analysis identified several fatty acid oxidation (FAO) enzymes as proximity interactors including CPT2, ACADVL, ACADM, ECHI, HADH, and ACAA2. To corroborate these findings in LSCs, we performed RNA-sequencing upon SIRT3 inhibition in primary LSCs derived from three primary AML specimens. Gene set enrichment analysis revealed a significant decrease in OXPHOS and FAO gene signatures upon SIRT3 inhibition. Importantly, FAO is known to support OXPHOS which suggests that SIRT3 may regulate OXPHOS through mediation of FAO. To determine if protein interactions and gene expression changes result in functional changes in mitochondrial biology, we measured OXPHOS and mitochondrial ATP production in LSCs upon SIRT3 perturbation in LSCs using a seahorse assay. This analysis revealed that SIRT3 perturbation resulted in decreased OXPHOS and ATP production. To determine if SIRT3 regulates OXPHOS via FAO mediation, we performed mass spectrometry-based lipidomic and metabolic interrogation on LSCs from three AML samples treated with YC8-02, which showed an accumulation of fatty acid upon SIRT3 inhibition. To interrogate fatty acid metabolism further, we performed stable isotope tracing analysis using 13C16-palmitate on LSC enriched from three patient specimens upon SIRT3 inhibition. Tracing studies revealed a reduction in 13C enrichment in tricarboxylic acid (TCA) cycle metabolites in the YC8-02-treated LSCs (Fig. 1B), suggesting an impairment to fatty acid utilization upon SIRT3 inhibition. Decreasing availability of TCA products impairs OXPHOS and ultimately causes cell death. Importantly, viability of LSCs was partially rescued through addition of the TCA intermediate α-ketoglutarate, demonstrating reduced FAO to be an essential part of the mechanism by which SIRT3 targets LSCs. In addition to decreasing OXPHOS, the accumulation of fatty acids should induce cell death by causing lipotoxicity. However, phenocopy experiments revealed that LSCs are protected from lipotoxicity unlike AML blasts or AML cell lines. Lipidomic analysis suggested that fatty acids may be converted to cholesterol esters, which can then be stored or excreted from cells, specifically in LSCs and not in AML cell lines. Therefore, we postulated that targeting cholesterol homeostasis using dipyridamole would enhance the effect of SIRT3 inhibition (Esquejo et al., 2021). The combination treatment significantly reduced the viability of LSCs compared to individual agents, indicating that co-inhibition of SIRT3 and cholesterol homeostasis enhances the toxic effects of FAO inhibition on LSCs. In all, we identify SIRT3 as a key regulator of LSC survival, through the mediation of lipid metabolism. We further demonstrate that LSCs are tolerant to FAO inhibition which can be overcome by concurrent inhibition of FAO and cholesterol metabolism, which has the potential to lead to improved treatment response. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Structural Surfaceomics Reveals an AML-Specific Conformation of Integrin-β2 As an Immunotherapeutic Target

Background and Significance: Cellular therapies have led to immense excitement in cancer care, with CAR-T cells as the canonical example. However, the clinical reach of these "living drugs" is still limited. A major hurdle in this regard is the lack of cancer-specific surface markers. Here, we explored the hypothesis that tumor surface antigens may harbor cancer-specific protein conformations, invisible to standard target discovery strategies focusing on gene or protein expression alone, that may create selective immunotherapy targets. While one previous example existed in the literature (Hosen et al., Nat Med (2017) 23:1436), this was a serendipitous discovery not intended to find conformation-specific antigens. We thus aimed to develop a technology that could identify conformation-specific tumor antigens in a broad, systematic manner. We first focused on finding such antigens in acute myeloid leukemia (AML), given that current leading immunotherapy targets (e.g. CD123, CD33, CLEC12A) are also expressed widely on normal hematopoietic cells, leading to toxicities and an urgent need for targets with greater tumor specificity. Results: Toward unbiased discovery of tumor-specific, conformationally-based cancer antigens, we developed a proteomic approach called "structural surfaceomics". This method sequentially integrates cross-linking mass spectrometry (XL-MS) with glycoprotein cell surface capture (CSC) to enable low-resolution structural profiling of hundreds of cell surface proteins in parallel. We used two different XL-MS crosslinkers: the mass spec-cleavable DSSO and the non-cleavable PhoX, the latter including a phosphonate handle for peptide enrichment. In application to Nomo-1 AML cells, these strategies yielded complementary information, in total resulting in 2,390 crosslinks on 250 membrane-localized proteins. Analyzing these data in comparison to Protein Data Bank structures, we specifically focused on a pattern of crosslinks that suggested the active conformation of integrin-β2 may be present on AML (Figure 1a). While integrin-β2 is known to be widely expressed on hematopoetic cells (and confirmed to be absent on non-hematopoietic tissues, based on our analysis of Human Protein Atlas single-cell RNA-seq data), the vast majority of normal blood cells are thought to harbor this antigen in the closed, resting conformation. In contrast, we validated that integrin-β2 appears to be constitutively present in the active conformation on AML cell lines as well as primary patient blasts, suggesting a possible therapeutic index. Indeed, total integrin-β2, but not the active conformation, was also found on normal hematopoietic stem and progenitor cells (HSPCs). Toward therapeutically targeting the active conformation of integrin-β2, we used a humanized antibody fragment library displayed on phage to identify a conformation-selective binder. We reconstructed this binder in CAR-T format (aITGB2 CAR-T) to develop a novel proof-of-principle AML therapy, finding potent cytotoxicity against AML cell lines in vitro as well as PDX and cell line models of AML implanted in NSG mice (Figure 1b). Further functional experiments using human blood mononuclear cells and AML cell lines supported that our binder did not interact with total integrin-β2 but was selective for the active conformation. This promising safety profile was supported by experiments in NSG-SGM3 mice with a reconstituted human immune system, where aITGB2 CAR-T had no impact on human CD45+ cells but significant depletion was seen with anti-CD33 CAR-T. Our recombinant antibody binder also was cross-reactive with murine active integrin-β2; complete blood count profiling of mice treated with aITGB2 CAR-T demonstrated no peripheral blood cell depletion. Conclusion: With this study, we demonstrate a systematic approach for identification of conformation-specific cancer antigens at the cell surface. The successful engineering of aITGB2 CAR-Ts demonstrates an encouraging proof-of-principle therapy for AML, with promising efficacy and a favorable safety profile compared to current leading targets. These results underscore the potential of "structural surfaceomics" to expand the toolkit of target discovery in cancer, with possible applications in other diseases, while motivating further preclinical evaluation of aITGB2 CAR-Ts in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Systematic Evaluation of AML-Associated Antigens Identifies Novel Anti-U5 snRNP200 Therapeutic Antibodies for the Treatment of AML

Numerous antibody-based immunotherapies are in development for AML including antibodies targeting CD47, CD70, CD123, CD33, and TIM3. However, systematic evaluation of the distribution of AML-associated antigens in combination with Fc receptors and the immune microenvironment required for antibody-mediated therapeutic responses have been limited. Additionally, efforts to maximize antibody Fc receptor engagement on immune effector cells in AML patients via Fc region engineering have not been explored. Here we applied a custom 36-color flow cytometry panel tailored for AML patient bone marrow along with proteogenomic assessment via CITE-seq to simultaneously assess known and novel antibody target expression on AML cells, immune microenvironment, and Fc receptor expression across immune subsets. Application to samples from 7 age-matched healthy donors and 51 newly diagnosed AML patients allowed for unbiased identification of the malignant blast population via UMAP projection. Quantification of differential antigen expression on blasts and leukemic stem cells versus normal hematopoietic stem and progenitor cell (HSPC) subsets, identified that out of 9 AML putative antibody targets (CD123, TIM3, CD33, CD47, CD90, CD38, CD25, CD70, U5 snRNP200), only U5 snRNP200 is expressed on blasts but not normal HSPCs (Fig. A). Characterization of the distribution of Fc receptors revealed that AML patients exhibit reduced cell-surface activating Fc receptors (CD16, CD32A) on NK and non-classical monocytes with concomitant increases in inhibitory (CD32B) Fc receptors on monocytes and B-cells. Moreover, AML patients had fewer classical monocytes and type 2 conventional dendritic (cDC2) cells than normal age-match control marrow. Given the unique cell surface expression of U5 snRNP200 on AML blasts compared with normal HSPCs, we deeply interrogated the distribution of this marker throughout normal and malignant hematopoiesis. While U5 snRNP200 expression was not seen on normal HSPCs, U5 snRNP200 was present on mature B-cells and a subset of NK cells in normal adult human and mouse hematopoietic cells. U5 snRNP200 is an RNA helicase which is an essential conserved component of the spliceosome. Its known function is limited to the nucleus and it is therefore unexpected that it would be expressed on the cell surface. To rigorously confirm expression of U5 snRNP200 on the cell membrane, we knocked in a HaloTag at the N-terminus of the locus encoding U5 snRNP200 within K562 cells and traced expression of tagged endogenous U5 snRNP200 protein across cellular compartments. This clearly revealed cell surface localization of the full-length protein on the surface of AML cells. We next conducted genome-wide CRISPR screens in two human AML cell lines to identify regulators of surface U5 snRNP200 expression. In both cell lines, the top gene required for cell surface U5 snRNP200 expression was CD32A. CRISPR knockout of CD32A in U937 cells reduced U5 snRNP200 surface expression, while restoration of CD32A rescued U5 snRNP200 surface expression. AML-associated expression of U5 snRNP200 was present on a number of syngeneic immunocompetent mouse models of AML including MLL-AF9, RN2 (MLL-AF9 + NRAS G12D), transgenic EVI1 rearranged/SF3B1K700E mutant, and Tet2/Zrsr2 double knockout mice. To test the therapeutic potential of targeting cell surface U5 snRNP200, we engineered several anti-murine U5 snRNP200 antibody variants featuring modified Fc regions to assess the possible roles of engagement of Fc receptors on immune cell subsets to enhance AML cell killing. In both the RN2 and EVI1/SF3B1 mutant models, anti-U5 snRNP200 antibodies featuring the Fc region modified to engage activating Fc receptors and induce antibody-dependent cellular cytotoxicity (IgG2a) provided superior survival benefit compared to IgG1 or IgG2b U5 snRNP200 antibodies or a mutant IgG1 antibody (IgG1 D265A) which does not bind Fc receptors (Fig. B). These data provide a high-density roadmap of the distribution of known and novel AML-associated antigens together with the Fc receptor distribution and immune microenvironment in AML. The results motivated discovery of a novel antibody-therapeutic targeting aberrant cell-surface U5 snRNP200 and research into mechanisms for cell surface trafficking of U5 snRNP200. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Decreased Embryonic Splicing Repressor Expression Enhances Splicing Modulator Sensitivity in Pediatric Acute Myeloid Leukemia Stem Cells

Adults with myeloid malignancies frequently harbor mutations in splicing regulatory genes, such as SRSF2, U2AF1, and SF3B1. In pediatric AML (pAML), splicing factor mutations are relatively infrequent in bulk populations of cells. However, by performing whole exome sequencing, single cell proteogenomics (Tapestri®) as well as whole transcriptome RNA sequencing (RNA-seq) analyses of FACS-purified hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs), we discovered an increased frequency of exon skipping, typical of embryonic splicing patterns, in pAML HSCs and HPCs. By employing single cell proteogenomics (Tapestri®) analysis, we discovered SF3B1 splicing factor mutations at the single cell in pAML CD34+ cells that were not readily detectable by whole exome sequencing in bulk populations thereby suggesting that clonal expansion of minor clones may fuel leukemic stem cell (LSC) generation. Further RNA-seq analysis demonstrated that both pAML HSCs and HPCs harbored decreased expression of a repressor of embryonic splicing, RNA-binding Fox protein 2, RBFOX2(also known as RBM9). By employing, rMATS, an algorithm that detects splicing events, we discovered differential splicing of genes involved in cell cycle regulation, DNA repair and chromatin organization in pAML more than in non-leukemic (NL) samples. In addition, differentially spliced RBFOX2 target genes were found to be involved in mRNA processing. In pAML HSCs, we observed differential splicing of THOC7, which regulates mRNA 3' end processing while in pAML HPCs, we uncovered a cluster of splicing regulators, such as SRSF7, SRSF11 and U2AF2. Thus, pAML is typified by widespread splicing deregulation in the setting of RBFOX2 downregulation. Previous reports suggest that splicing deregulation represents a therapeutic vulnerability in a broad array of malignancies. Thus, we examined the response of pAML cell lines in vitro and in lentiviral splicing reporter humanized AML mouse models to Rebecsinib (17S-FD-895; Crews et al, Cell Stem Cell, 2016), a small molecule splicing modulator that is currently undergoing IND enabling studies. In these studies, Rebecsinib treatment reduced pAML cell survival at doses that altered splicing reporter activity and spared normal hematopoietic stem and progenitor cells (HSPCs). Notably, shRNA knockdown of RBFOX2 in cord blood CD34+ HSPCs phenocopied aspects of pAML in that it significantly increased clonogenic capacity and sensitivity to Rebecsinib in replating assays. In summary, decreased RBFOX2 expression and reversion to an embryonic splicing program typified by differential exon usage in pAML HSPCs was associated with increased sensitivity to selective splicing modulation and may represent a new therapeutic paradigm for pediatric patients with relapsed or refractory AML.

TIF1β Enhances Self-Renewal Capacity of BCR-ABL Leukemic Stem Cells and Inhibits the Myeloid Differentiation

Chronic myeloid leukemia (CML), characterized by expression of p210-BCR-ABL, is a clonal myeloproliferative disease initiated by malignant transformation of hematopoietic stem cells (HSC). Blastic crisis phase (BC) of CML that shows expansion of therapy-refractory and differentiation-arrested blasts, remains a therapeutic challenge. Transcription intermediary factor TIF1β/TRIM28/KAP1 modulates chromatin structure to repress or activate transcription of target genes. Tif1β is essential for the proliferation and differentiation of normal hematopoietic stem cells, but the role of Tif1β in myeloid leukemia is unclear. Recently, high expression of TIF1β was correlated with poor prognosis in patients with various types of cancer. By analyzing datasets of gene expression in bone marrow (BM) cells in CML patients, we found that TIF1β expression levels were robustly elevated in CML cells at the BC stage. Thus, we hypothesized that TIF1β functions as an oncogene for the development of BCR-ABL leukemia. To determine whether TIF1β promoted the development of BCR-ABL-induced myeloid leukemia in vivo, we generated BCR-ABL;Tif1βf/f;Cre-ERT2 compound mice by crossing Rosa26 locus BCR-ABL conditional knock-in mice and Tif1βf/f;Cre-ERT2 conditional KO mice. We transplanted BM cells isolated from Cre-ERT2 (WT), Tif1βf/f;Cre-ERT2 (Tif1β KO), BCR-ABL;Cre-ERT2 (BCR-ABL), and BCR-ABL;Tif1βf/f;Cre-ERT2 (BCR-ABL;Tif1β KO) mice into lethally irradiated CD45.1+ WT recipient mice, and injected tamoxifen 4 weeks after BM transplantation. While BCR-ABL KI mice showed rapid expansion of leukemic blast cells and died in a short period of time, the deletion of Tif1β gene resulted in a significantly longer survival with reduced tumor burden in BM, liver and spleen in BCR-ABL KI mice. BCR-ABL KI mice mostly developed myeloid leukemia, in contrast, BCR-ABL;Tif1β KO mice dominantly showed B-cell leukemia or bone marrow failure diseases. A subset of BCR-ABL;Tif1β KO mice survived without development of lethal leukemia in the primary and the secondary transplantation settings, indicating that TIF1β maintained the self-renewal capacity of leukemic stem cells (LSCs) but also controlled the myeloid/lymphoid lineage commitment. To elucidate the mechanisms underlying the impaired development of leukemia in BCR-ABL;Tif1βKO mice, we performed RNA sequencing analyses of HSCs isolated from WT, Tif1β KO, BCR-ABL, BCR-ABL;Tif1βKO mice. Gene set enrichment analysis revealed that the loss of Tif1β repressed BCR-ABL-induced activation of proliferative stem cell signatures, but increased expression of myeloid differentiation genes including PU.1 gene in HSCs. We found that BCR-ABL;Tif1βKO HSCs reduced expression levels of Myc and Myc-target genes including Fra1 transcription factor (TF), which was known to promote tumor cell proliferation. Notably, human CML-CP and CML-BC CD34+ cells markedly increased expression levels of MYC and FRA1 genes. To clarify how the Tif1β protein controlled self-renewal of LSCs, we performed ATAC-sequencing in murine HSCs and TIF1β-ChIP-sequencing using BCR-ABL-expressing human leukemia cells. ATAC-seq revealed that BCR-ABL KI HSCs showed enrichments of binding motif of Fra1 in open chromatin and that of PU.1 in closed chromatin, compared to WT HSCs, while BCR-ABL;Tif1βKO HSCs cancelled those enrichments of binding motifs of TFs. TIF1β-ChIP-seq revealed that the TIF1β protein directly bound to a promoter region of the FRA1 gene. TIF1β binding sites were enriched with canonical PU.1- and JUN/FRA1-binding motifs. We also found that treatment with imatinib decreased enrichment of FRA1-binding motif among TIFβ-binding regions in BCR-ABL+ cells, indicating that TIFβ opened chromatin accessibility to activate expression of and transcriptional function of the Fra1 gene in a BCR-ABL dependent manner, but repressed the accessibility of myeloid differentiation regulator TFs. Furthermore, knockdown of Fra1 significantly impaired the proliferation of BCR-ABL stem cells in an in vitro condition. In conclusion, we demonstrate that the Tif1β gene was required for the propagation of BCR-ABL-induced myeloid leukemia stem cell in mice. Tif1β enhanced chromatin accessibility of Fra1 TF to promote transcription of LSC signature genes, but also reduced chromatin accessibility at the PU.1-binding motif, resulting in the enhanced proliferation and limited differentiation of LSCs.