Murine Hematopoietic Stem Cells Research Articles

Kinase-inactivated CDK6 preserves the long-term functionality of adult hematopoietic stem cells

AbstractHematopoietic stem cells (HSCs) are characterized by the ability to self-renew and to replenish the hematopoietic system. The cell-cycle kinase cyclin-dependent kinase 6 (CDK6) regulates transcription, whereby it has both kinase-dependent and kinase-independent functions. Herein, we describe the complex role of CDK6, balancing quiescence, proliferation, self-renewal, and differentiation in activated HSCs. Mouse HSCs expressing kinase-inactivated CDK6 show enhanced long-term repopulation and homing, whereas HSCs lacking CDK6 have impaired functionality. The transcriptomes of basal and serially transplanted HSCs expressing kinase-inactivated CDK6 exhibit an expression pattern dominated by HSC quiescence and self-renewal, supporting a concept, in which MAZ and NFY-A are critical CDK6 interactors. Pharmacologic kinase inhibition with a clinically used CDK4/6 inhibitor in murine and human HSCs validated our findings and resulted in increased repopulation capability and enhanced stemness. Our findings highlight a kinase-independent role of CDK6 in long-term HSC functionality. CDK6 kinase inhibition represents a possible strategy to improve HSC fitness.

Context-dependent modification of PFKFB3 in hematopoietic stem cells promotes anaerobic glycolysis and ensures stress hematopoiesis

Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in murine HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of ATP levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells.

Abstract 5598: Combined loss of the chromosome 7 genes CUX1 and EZH2 promotes chemotherapy resistance

Abstract Monosomy 7 (-7) or del(7q) are recurrent in high-risk myeloid neoplasms, including up to 50% of myeloid neoplasms arising after prior exposure to chemotherapy and/or radiation. Although putative chromosome 7 tumor suppressor genes have been identified, the effects of combinatorial 7q gene loss remain unclear. A barrier to understanding the pathogenesis of -7/del(7q) is the challenge of modeling aneuploidy in animal models. To address this knowledge gap, we established an in vivo model of del(7q) clonal hematopoiesis and drug resistance using multiplex CRISPR-Cas9 to simultaneously target four 7q genes (Cux1, Ezh2, Kmt2c, and Kmt2e) in murine hematopoietic stem cells. After chemotherapy exposure, we observe significant myeloid expansion of clones edited for both Cux1 and Ezh2. Compared to the transcriptomes of control or single gene edited cells, Cux1;Ezh2-deficient cells fail to induce DNA damage response pathways after genotoxic stress. Cux1;Ezh2-deficient cells also display reduced deposition of γH2AX after DNA damage, as well as persistent, unrepaired DNA breaks, indicating perturbations to DNA damage recognition and repair. Collectively, our data support the concept of 7q as a contiguous gene syndrome region, in which combined loss of multiple genes drives drug resistance and disease development. This work reveals a genetic interaction between CUX1 and EZH2, and sheds light on how -7/del(7q) contributes to the development and inherent drug resistance characteristic of high-risk myeloid disease. Further, our CRISPR-based approach may serve as a framework for interrogating other recurrent aneuploid events in cancer. Citation Format: Matthew R. Jotte, Angela Stoddart, Tanner C. Martinez, Yuqing Xue, Molly K. Imgruet, Megan E. McNerney. Combined loss of the chromosome 7 genes CUX1 and EZH2 promotes chemotherapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5598.

Abstract 5589: CBFB::MYH11 expression activates Trp53 in myeloid cells, which inhibits cell growth and sensitizes cells to cytarabine

Abstract CBFB::MYH11 acute myeloid leukemia (AML) is a subset of Core Binding Factor AML initiated by an oncofusion in which Core Binding Factor Beta (CBFB) is fused to the C-terminus of smooth muscle myosin heavy chain 11 (MYH11). CBFB::MYH11 AML has a high cure rate with conventional anthracycline/cytarabine induction and high-dose cytarabine consolidation in patients under the age of 60, and remains chemosensitive after multiple relapses; the mechanisms underlying chemosensitivity are unknown. TP53 is a key regulator of DNA damage response and apoptosis, and TP53-mutated AML is often chemoresistant. Large sequencing studies have revealed only rare co-occurrence of TP53 mutations with CBFB::MYH11. We initially used CBFB::MYH11 as a negative control for an unrelated study of TP53 function and unexpectedly found striking synergy. While expression of CBFB::MYH11 in WT murine hematopoietic stem and progenitor cells (HSPCs) induced a low-penetrance (~20%), long latency (~6 months) myelomonocytic AML, expression of CBFB::MYH11 in Trp53-/- HSPCs induced AML with 100% penetrance and a median latency of 8 weeks. CBFB::MYH11-induced AMLs arising in Trp53-/- cells were similar by morphology and flow cytometry phenotype. We validated these findings in a second model, using a conditional Trp53flox/flox x Vav1-Cre model in which Trp53 is inactivated in HSPCs, with nearly identical results. Trp53-/- cells transduced with Empty Vector did not develop AML, but did develop T cell lymphoma with a median latency of 16 weeks, similar to prior studies. Trp53 deficiency also cooperated with RUNX1::RUNX1T1, the other Core Binding Factor oncofusion, to induce a high-penetrance (100%) and short latency AML (median latency 10 weeks); consistent with prior data, RUNX1::RUNX1T1 expression alone in Trp53 WT cells was insufficient to induce AML. To determine whether Trp53 influences the chemosensitivity of CBFB::MYH11 expressing cells, we performed cytarabine dose-response experiments. While CBFB::MYH11-transduced WT HSPCs were very sensitive to cytarabine (relative to EV), sensitivity was reduced in CBFB::MYH11-transduced Trp53-/- cells, suggesting that CBFB::MYH11-induced chemosensitivity is, at least in part, mediated by the TP53 pathway. To determine whether CBFB::MYH11 expression results in upregulation of key TP53 pathway genes, we transduced WT HSPCs with EV or CBFB::MYH11, transplanted cells into WT mice, harvested bone marrow at 3 months, and performed RNA-Seq. Key TP53 pathway genes were upregulated, including Bbc3/Puma, Bax, Cdkn1a/p21, and Trp53 (all 2.6-4.6-fold, p≤0.05). These data suggest that the chemosensitivity and the favorable prognosis of CBFB::MYH11 AML may be due to the direct activation of the TP53 pathway by CBFB::MYH11. Similar studies are underway with RUNX1::RUNX1T1-transduced cells to determine whether TP53 is relevant for the chemosensitivity of that CBF AML as well. Citation Format: Ryan B. Day, Julia A. Hickman, Casey D. Katerndahl, Sai Mukund Ramakrishnan, Christopher A. Miller, Timothy J. Ley. CBFB::MYH11 expression activates Trp53 in myeloid cells, which inhibits cell growth and sensitizes cells to cytarabine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5589.

Deep learning-based predictive classification of functional subpopulations of hematopoietic stem cells and multipotent progenitors.

Hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) play a pivotal role in maintaining lifelong hematopoiesis. The distinction between stem cells and other progenitors, as well as the assessment of their functions, has long been a central focus in stem cell research. In recent years, deep learning has emerged as a powerful tool for cell image analysis and classification/prediction. In this study, we explored the feasibility of employing deep learning techniques to differentiate murine HSCs and MPPs based solely on their morphology, as observed through light microscopy (DIC) images. After rigorous training and validation using extensive image datasets, we successfully developed a three-class classifier, referred to as the LSM model, capable of reliably distinguishing long-term HSCs, short-term HSCs, and MPPs. The LSM model extracts intrinsic morphological features unique to different cell types, irrespective of the methods used for cell identification and isolation, such as surface markers or intracellular GFP markers. Furthermore, employing the same deep learning framework, we created a two-class classifier that effectively discriminates between aged HSCs and young HSCs. This discovery is particularly significant as both cell types share identical surface markers yet serve distinct functions. This classifier holds the potential to offer a novel, rapid, and efficient means of assessing the functional states of HSCs, thus obviating the need for time-consuming transplantation experiments. Our study represents the pioneering use of deep learning to differentiate HSCs and MPPs under steady-state conditions. This novel and robust deep learning-based platform will provide a basis for the future development of a new generation stem cell identification and separation system. It may also provide new insight into the molecular mechanisms underlying stem cell self-renewal.

Ex vivo expansion potential of murine hematopoietic stem cells is a rare property only partially predicted by phenotype.

The scarcity of hematopoietic stem cells (HSCs) restricts their use in both clinical settings and experimental research. Here, we examined a recently developed method for expanding rigorously purified murine HSCs ex vivo. After 3 weeks of culture, only 0.1% of cells exhibited the input HSC phenotype, but these accounted for almost all functional long-term HSC activity. Input HSCs displayed varying potential for ex vivo self-renewal, with alternative outcomes revealed by single-cell multimodal RNA and ATAC sequencing profiling. While most HSC progeny offered only transient in vivo reconstitution, these cells efficiently rescued mice from lethal myeloablation. The amplification of functional HSC activity allowed for long-term multilineage engraftment in unconditioned hosts that associated with a return of HSCs to quiescence. Thereby, our findings identify several key considerations for ex vivo HSC expansion, with major implications also for assessment of normal HSC activity.

Ex vivo expansion potential of murine hematopoietic stem cells is a rare property only partially predicted by phenotype

The scarcity of hematopoietic stem cells (HSCs) restricts their use in both clinical settings and experimental research. Here, we examined a recently developed method for expanding rigorously purified murine HSCs ex vivo. After 3 weeks of culture, only 0.1% of cells exhibited the input HSC phenotype, but these accounted for almost all functional long-term HSC activity. Input HSCs displayed varying potential for ex vivo self-renewal, with alternative outcomes revealed by single-cell multimodal RNA and ATAC sequencing profiling. While most HSC progeny offered only transient in vivo reconstitution, these cells efficiently rescued mice from lethal myeloablation. The amplification of functional HSC activity allowed for long-term multilineage engraftment in unconditioned hosts that associated with a return of HSCs to quiescence. Thereby, our findings identify several key considerations for ex vivo HSC expansion, with major implications also for assessment of normal HSC activity.

Abcb10 regulates murine hematopoietic stem cell potential and erythroid differentiation

Erythropoiesis in the adult bone marrow relies on mitochondrial membrane transporters to facilitate heme and hemoglobin production. Erythrocytes in the bone marrow are produced although the differentiation of erythroid progenitor cells that originate from hematopoietic stem cells (HSCs). Whether and how mitochondria transporters potentiate HSCs and affect their differentiation toward erythroid lineage remains unclear. Here, we show that the ATP-binding cassette (ABC) transporter 10 (Abcb10), located on the inner mitochondrial membrane, is essential for HSC maintenance and erythroid-lineage differentiation. Induced deletion of Abcb10 in adult mice significantly increased erythroid progenitor cell and decreased HSC number within the bone marrow (BM). Functionally, Abcb10-deficient HSCs exhibited significant decreases in stem cell potential but with a skew toward erythroid-lineage differentiation. Mechanistically, deletion of Abcb10 rendered HSCs with excess mitochondrial iron accumulation and oxidative stress yet without alteration in mitochondrial bioenergetic function. However, impaired hematopoiesis could not be rescued through the in vivo administration of a mitochondrial iron chelator or antioxidant to Abcb10-deficient mice. Abcb10-mediated mitochondrial iron transfer is thus pivotal for the regulation of physiologic HSC potential and erythroid-lineage differentiation.

Ultraviolet Light-Emitting Diode Radiation Kills Leukemia Cells Without Affecting Hematopoiesis of Hematopoietic Stem Cells in Mice.

The eradication of leukemia cells while sparing hematopoietic stem cells in the graft before autologous hematopoietic stem cell transplant is critical to prevention of leukemia relapse. Proliferating cells have been shown to be more prone to apoptosis than differentiated cells in response to ultraviolet radiation; however, whether leukemia cells are more sensitive to ultraviolet LED radiation than hematopoietic stem cells remains unclear. We compared the in vitro responses between murine leukemia L1210 cells and murine hematopoietic stem cells to 280-nm ultraviolet LED radiation. We also investigated the effects of ultraviolet LED radiation on the tumorigenic and metastatic capacity of L1210 cells and hematopoietic stem cell hematopoiesis in a mouse model of hematopoietic stem cell transplant. L1210 cells were more sensitive to ultraviolet LED radiation than hematopoietic stem cells in vitro, as evidenced by significantly reduced colony formation rates and cell proliferation rates, along with remarkably increased apoptosis rates in L1210 cells. Compared with corresponding unirradiated cells, ultraviolet LED-irradiated L1210 cells failed to generate palpable tumors in mice, whereas ultraviolet LED-irradiated bone marrow cells restored hematopoiesis in vivo. Furthermore, transplant with an irradiated mixture of L1210 cells and bone marrow cells showed later onset of leukemia, milder leukemic infiltration, and prolonged survival in mice, compared with unirradiated cell transplant. Our results suggest that ultraviolet LED radiation can suppress the proliferative and tumorigenic abilities of leukemia cells without reducing the hematopoietic reconstitution capacity of hematopoietic stem cells, serving as a promising approach to kill leukemia cells in autograft before autologous hematopoietic stem cell transplant.

Chromatin accessibility and cell cycle progression are controlled by the HDAC-associated Sin3B protein in murine hematopoietic stem cells

BackgroundBlood homeostasis requires the daily production of millions of terminally differentiated effector cells that all originate from hematopoietic stem cells (HSCs). HSCs are rare and exhibit unique self-renewal and multipotent properties, which depend on their ability to maintain quiescence through ill-defined processes. Defective control of cell cycle progression can eventually lead to bone marrow failure or malignancy. In particular, the molecular mechanisms tying cell cycle re-entry to cell fate commitment in HSCs remain elusive. Previous studies have identified chromatin coordination as a key regulator of differentiation in embryonic stem cells.ResultsHere, we utilized genetic inactivation of the chromatin-associated Sin3B protein to manipulate cell cycle control and found dysregulated chromatin accessibility and cell cycle progression in HSCs. Single cell transcriptional profiling of hematopoietic stem and progenitor cells (HSPCs) inactivated for Sin3B reveals aberrant progression through the G1 phase of the cell cycle, which correlates with the engagement of specific signaling pathways, including aberrant expression of cell adhesion molecules and the interferon signaling program in LT-HSCs. In addition, we uncover the Sin3B-dependent accessibility of genomic elements controlling HSC differentiation, which points to cell cycle progression possibly dictating the priming of HSCs for differentiation.ConclusionsOur findings provide new insights into controlled cell cycle progression as a potential regulator of HSC lineage commitment through the modulation of chromatin features.

Age-related noncanonical TRMT6-TRMT61A signaling impairs hematopoietic stem cells.

Aged hematopoietic stem cells (HSCs) exhibit compromised reconstitution capacity and differentiation bias toward myeloid lineages. However, the molecular mechanism behind HSC aging remains largely unknown. In this study, we observed that RNA N1-methyladenosine-generating methyltransferase TRMT6-TRMT61A complex is increased in aged murine HSCs due to aging-declined CRL4DCAF1-mediated ubiquitination degradation signaling. Unexpectedly, no difference of tRNA N1-methyladenosine methylome is observed between young and aged hematopoietic stem and progenitor cells, suggesting a noncanonical role of the TRMT6-TRMT61A complex in the HSC aging process. Further investigation revealed that enforced TRMT6-TRMT61A impairs HSCs through 3'-tiRNA-Leu-CAG and subsequent RIPK1-RIPK3-MLKL-mediated necroptosis cascade. Deficiency of necroptosis ameliorates the self-renewal capacity of HSCs and counters the physiologically deleterious effect of enforced TRMT6-TRMT61A on HSCs. Together, our work uncovers a nonclassical role for the TRMT6-TRMT61A complex in HSC aging and highlights a therapeutic target.

Genetic and Pharmacological Interruption of the SIX1/EYA2 Axis Impairs Leukemia Proliferation

Background: The CALM-AF10 translocation is found in 5-10% of T-cell acute lymphoblastic leukemias (T-ALL), and a subset of acute myeloid leukemias (AML). CALM-AF10 leukemias are characterized by elevated expression of proleukemic HOXA genes. Since HOXA genes are difficult to target, we sought to identify non- HOXA effector genes through RNA sequencing and Microarray analyses, and showed that SIX1 expression is increased in CALM-AF10 leukemia cells. Like HOXA genes, SIX1 is a homeobox gene involved in cell proliferation, embryogenesis, and apoptosis. SIX1 interacts with the cofactor Eyes Absent 2 (EYA2), a protein phosphatase, to transcriptionally activate developmental genes, the TGFβ pathway, and the epithelial-mesenchymal transition (EMT). SIX1 expression is elevated during development but decreases with maturation. Intriguingly, overexpression of SIX1 has been observed in numerous malignancies including breast cancer, ovarian cancer, gliomas and esophageal cancer, and SIX1 is involved in accelerating the epithelial mesenchymal transition (EMT) and metastasis. Knockdown of either SIX1 or EYA2 has been shown to reverse these effects, and two novel inhibitors - one of the SIX1/EYA2 complex (Compound 8430, “8430”) and one of the EYA2 phosphatase (Compound 9987, “9987”) - have replicated the effects of SIX1 or EYA2 knockdown. Methods: SIX1 gene and protein expression were assessed in leukemia cell lines via RT-qPCR and Western Blot. SIX1 expression vectors were transduced into fetal liver hematopoietic stem cells (HSC) and immortalization was assessed using colony assays. CALM-AF10 leukemia cells lines were derived from irradiated mice transplanted with HSC retrovirally transfected with CALM-AF10 plasmids created in our lab. The established leukemia cell lines Jurkat, MOLT16, OCI-M2, TF-1, and SHI-1 were obtained from ATCC or DSMZ. shRNAs targeting SIX1 were lentivirally transduced into CALM-AF10 and SHI1leukemia cell lines. Cell-Titer-Glo assays and liquid culture were used to assess the effects of shRNAs, 8430, or 9987 on cell proliferation and cell growth. SIX1 gene expression in leukemias was determined from The Cancer Dependency Map from The Broad Institute using R. Results: We first validated RNAseq and Microarray studies, showing that CALM-AF10 induces SIX1 expression in murine fibroblasts and murine HSC, and localizes to the SIX1 locus. Furthermore, SIX1 overexpression was sufficient to immortalize murine fetal liver HSC (FL-mHSCs), implying a role in leukemic transformation. Multiple murine derived CALM-AF10 cell lines displayed increased SIX1 expression compared to fresh murine bone marrow and isolated HSCs. shRNA knockdown of SIX1 decreased viability of murine CALM-AF10 cells in culture. In addition, both the SIX1/EYA2 complex inhibitor (8430) and the EYA2 inhibitor (9987) decreased CALM-AF10 leukemia cell proliferation in vitro. To expand the relevance of SIX1 to other leukemias, we then queried The Cancer Dependency Map from the Broad Institute and identified other leukemia cell lines with increased SIX1 expression, including Jurkat and Molt16 (T-ALL), and SHI-1, OCIM2, and TF-1 (AML). SIX1 expression in these cells was validated by immunoblot and shRNA knockdown of SIX1 slowed the proliferation of SHI-1 cells. Finally, the 8430 and 9987 inhibitors reduced the proliferation of Jurkat, OCI-M2, and SHI-1 cells at doses similar to those used in CALM-AF10 leukemias. Conclusions: We have identified involvement of the SIX1/EYA2 axis in several different leukemias, including T-ALL and AML. Furthermore, we have shown that inhibitors of the SIX1/EYA2 interaction and of EYA2 reduce the proliferation of SIX1 expressing leukemias, suggesting a potential new therapeutic target in these leukemias.

Srcap Plays an Essential Role in Normal Hematopoiesis

The incorporation of variant histones into chromatin has been revealed to be an important mechanism of epigenetic regulation. Deposition of H2A.Z, in particular, is known to mediate several essential cellular processes including chromatin organization, DNA damage response, and transcriptional regulation. Nonsense and frameshift mutations in the H2A.Z exchanger, SRCAP, have been recurrently identified in both clonal hematopoiesis and AML, suggesting the importance of this gene in both non-malignant hematopoiesis and leukemogenesis. However, the precise role of SRCAP and the impact of its dysregulation on hematopoiesis remain unknown. To investigate the role of Srcap in hematopoiesis, we generated a conditional Srcap loss of function murine model with loxP sites flanking exons 11-16. Cre-induced deletion of these exons results in a loss of the catalytic ATPase domain along with a frameshift within the remaining protein sequence. When Vav1-iCre +/-Srcap fl/+ and Srcap fl/fl mice are crossed, no Vav1-iCre +/-Srcap fl/fl pups are born, suggesting that hematopoietic-specific-loss of Srcap is embryonic lethal. At E14.5/15.5, Vav1-iCre +/-Srcap fl/fl embryos are paler than other genotypes ( Figure 1) with a reduced number of Ter119 + fetal liver cells. Interestingly, the decrease in Ter119 + cells upon Srcap loss occurs primarily in later stage erythroid progenitors, whereas the number of stage 1 erythroid progenitors is relatively well preserved, suggesting that loss of Srcap adversely impacts fetal erythroid maturation. Furthermore, Srcap loss impacts fetal liver myeloid maturation. While the number of granulocytes is unchanged in Srcap deficient embryos, they have a near complete absence of monocytes, monocyte-dendritic cell progenitors, and monocyte progenitors. In contrast to the changes observed within mature hematopoietic populations, phenotypic hematopoietic stem cells (HSCs) are maintained in Vav1-iCre +/-Srcap fl/fl fetal livers. However, their function is impaired as fetal liver HSCs fail to repopulate upon transplantation ( Figure 2). Importantly, adult HSCs isolated from Mx1-Cre +/-Srcap fl/flmice show a very similar phenotype in that they fail to maintain hematopoiesis when transplanted and treated with poly (I:C). In total, this data shows that Srcap is essential for the maintenance and proper function of both mature hematopoietic populations and hematopoietic progenitors with different lineages and developmental stages displaying varied sensitivities to Srcap loss. To investigate the cellular consequences of SRCAP loss, we knocked down SRCAP expression in hematopoietic cell lines using an inducible shRNA approach. In agreement with the results obtained from our murine model, SRCAP deficient MOLM13 cells show a baseline competitive growth disadvantage that becomes apparent within one week of culture. This is associated with increased γ-H2AX activity, suggesting enhanced DNA damage and consistent with the previously identified role of SRCAP in the cellular DNA damage response. Interestingly, murine fetal liver HSCs with loss of Srcap also exhibit increased γ-H2AX activity, and Srcap null fetal liver stage 1 erythroid progenitors display marked transcriptional upregulation of multiple p53 downstream genes. Our data demonstrate that, in hematopoietic populations, Srcap is an essential gene, with its complete loss resulting in enhanced cellular stress potentially due to increased DNA damage. In humans, nonsense and frameshift mutations in SRCAP appear to be heterozygous in nature, suggesting that the impact of SRCAP deficiency is dose dependent. Indeed, murine HSCs with heterozygous loss of Srcap maintain normal repopulating activity in both primary and secondary transplantation assays ( Figure 2). It remains to be determined how the intermediate deficiencies in SRCAP levels found in patients impact hematopoiesis and lead to clonal expansion and whether the impact of SRCAP loss on the cellular response to stress, including DNA damage, is mediated by its canonical function as a histone variant H2A.Z exchanger. These studies promise to reveal novel insights on the nature of H2A.Z-mediated epigenetic regulation in HSCs and on how its dysregulation may lead to leukemogenesis.

In Vivo Modeling of T-Cell Acute Lymphoblastic Leukemia Reveals Synergistic Oncogenic Pathways and Bcl11b Haploinsufficiency As a Potential Therapeutic Vulnerability

For decades, T-ALL models were based on retroviral overexpression of NOTCH1 in murine hematopoietic stem cells (HSCs). This induces aggressive T-ALL but does not reflect the genetic and clinical heterogeneity of the disease. Large-scale genomic analyses have mapped the detailed landscape of T-ALL driver events over recent years, but how these diverse oncogenic events interact in T-ALL pathogenesis and which vulnerabilities this creates remains incompletely explored. This is in part due to the lack of appropriate mouse models that recapitulate the complex disease biology and are suitable to identify and study potential therapeutic targets and interventions in a more realistic context. We modeled the genetic heterogeneity of T-ALL through in vivo multiplexing of gain-of-function and loss-of-function oncogenic events known to drive the disease. We developed a novel mouse strain that expresses Cre-recombinase and an inducible Cas9 only in the thymus ( LSL.Cas9 x Lck-cre) along with a lentiviral vector system into which T-ALL specific oncogenes are cloned in antisense flanked by LOX66/71 sites. Gain-of-function events, such as the overexpression of transcriptional regulators ( Tlx1, Tal1, or Lmo2) and signal transducers ( NRAS G12D, PIK3CD E1021K) were induced in developing thymocytes by Cre-mediated inversion together CRISPR-Cas9 editing of recurrent T-ALL loss-of-function drivers. This combinatorial approach allowed to probe more than 2000 possible oncogenic mutational combinations in vivo. Transplantation of HSCs from LSL.Cas9 x Lck-cre mice transduced with our vector system into sublethally irradiated mice led to T-ALL development with different morphology and disease phenotypes. Developing leukemia harbored reproducible phenotypes, covering very immature CD4-CD8-CD25+CD44+ T-ALL in the Tlx1_NRAS subgroup, in contrast to the classical cortical CD4+CD8+CD3- phenotype (Tlx1_PIK3CD subgroup), as well as mature single-positive CD4+CD3+ or CD8+CD3+ blasts (Tal1_Lmo2 subgroup). We identified Tlx1, Tal1, Lmo2, NRAS G12Dand PIK3CD E1021Kas the main driver of disease phenotype and gene expression profile, whereas the loss-of-function mutations contributed to and accelerated the onset of the disease. Tumor-derived cell lines could be easily established and kept their phenotypic characteristics and mutation patterns. The resulting T-ALL tumors contained up to eight different disease-relevant genetic alterations, thus recapitulating the genetic complexity in humans and overcoming the long latency for spontaneous mutations in less complex mouse models. Among these, co-evolution of insertions and deletions in Notch1, Cdkn2a, Bcl11b, and Pten were among the most frequently observed mutations in definitive T-ALL, whereas others, e.g., in Dnm2, Phf6, Etv6, and Lef1, occurred more randomly. While gene editing by CRISPR-Cas9 mostly resulted in frame-shift mutations in both alleles, Bcl11b exhibited a strong haploinsufficient phenotype with exactly one knock-out and one intact allele in each case examined. Following this observation, further suppression of Bcl11b by shRNAs in Bcl11b +/- cell lines derived from our tumor models demonstrated rapid induction of cell death, which was more intense in T-ALL cells compared to non-malignant thymocytes. In addition, re-analysis of CRISPR-based loss-of-function screens of human hematopoietic cell lines retrieved from the Cancer Dependency Map confirmed BCLL11B as a strong selective dependency in human T-ALL. In summary, we model subgroup-specific T-ALL transformation through conditional and multiplexed in vivo gene editing and oncogene overexpression, thus creating a resource of novel mouse models that recapitulate the genetic and biological heterogeneity of the disease. Using our resource, we identified Bcl11b as a strong and selective context-specific dependency in T-ALL. Although Bcl11b is a transcription factor, our results could serve as an starting point for novel therapies that interfere with Bcl11b function, such as molecular glue degraders or protein interaction inhibitors.

Single Cell Analysis of Genes Involved in Asymmetric Hematopoietic Stem Cell (HSC) Division in Chronic Myeloid Leukemia (CML) : Evidence of the Regulation of Tetraspanin CD63 during Leukemia Progression

Tyrosine kinase inhibitor (TKI) therapies have profoundly changed the natural history and survival of CML patients. However, the majority of the patients remain on long-lasting therapies and switch one TKI to another, either because of intolerance or resistance. All TKI cessation trials showed approximately 50% of leukemia recurrence rates even after several years of TKI therapies, suggesting the persistence of residual leukemic stem cells in patients in deep molecular response (DMR). The resistance and persistence of CML HSC are related to intrinsic (BCR-ABL independent survival) or extrinsic (leukemic niche ) mechanisms some of which have been extensively studied. However, there has been no data with regard to the role of asymmetric cell division pathways in the persistence and progression of CML stem cell clone. Indeed, During asymmetric division process of hematopoietic stem cells, some proteins are differently segregated between daughter HSCs such as endosome associated tetraspanin proteins (CD53, CD63) and mitochondria-associated fission regulator Dynamin related protein-1(DRP1). Similarly, it has been shown that asymmetric organelle inheritance can influence HSC decisions towards differentiation versus self-renewal. Interestingly, the tetraspanin CD63 is known to be involved in the quiescence of human and murine HSC via activation of TGF-beta pathway, CD63 hi cells exhibiting increased self-renewal potential after the asymmetric division with reduced differentiation potential. Materials and Methods We have analyzed the transcriptome performed in a cohort of CML patients in chronic phase (CP) and blast phase (BP) (GSE4170) as well as in a validation cohort of RNA-sequencing (GSE100026). Similarly, single cell transcriptome analysis of Lin- CD34+ CD38- CML progenitors (GSE76312) were included in the study evaluating the expression of genes involved in asymmetric cell division. We have then generated an asymmetric hematopoietic stem cell risk score using a panel of genes involved in the asymmetric division of HSCs and correlated these results with the transcriptome of the CML cohorts above. A pseudotime analysis was performed to evaluate single cell trajectories of the genes identified. Results To determine the potential role of asymmetric cell division markers in the progression of CML, we have analyzed the expression of several of these genes during CML progression in two independent transcriptome cohorts. The regulation concordance between the two distinct cohorts was principally observed on asymmetric molecules which were found to be down-regulated during blast crisis as compared to chronic phase. The regulation concordance between the two distinct cohorts was principally observed on asymmetric molecules down regulated during blast crisis as compared to chronic phase suggesting less of asymmetric properties during advanced phase of the disease. The down regulation in patients in blast crisis was confirmed at single cell level for the expression of SELL, CD63, NUMB, HK2 and LAMP2. CD63 expression has also been found to be of interest in CML chronic phase. Indeed, single cell neural network and deep learning in CP samples showed that the CD63 expression was the best asymmetric gene marker predicting a poor prognosis and this appeared to be induced by opposite regulation of HK2 and NUMB. In patients in CP-CML, the single cell trajectory reconstitution revealed the major regulatory role of CD63 highlighting a trajectory cluster implicating HSPB1, PIM2, ANXA5, LAMTOR1, CFL1, CD52, RAD52, MEIS1, PDIA3, all genes known to be involved also in hematopoietic malignancies such as myelodysplasia, AML and ALL. Conclusion We show here for the first time the identification of the expression of genes involved in the asymmetric division of CML HSC. Among these genes we identified the tetraspanin CD63 as being down-regulated during CML progression. The expression of the genes involved in the asymmetric HSC division should require further studies in CML, either to predict persistence of CML or progressive disease. Figure 1 : Single cell transcriptome of Lin- CD34+ CD38- CML progenitors in CP links CD63 expression to progressive disease. A/ Piechart of counts for chronic phase cells analyzed according to prognosis; B/ pseudotime transformation of single cell transcriptome according patient prognosis; C/ Pseudotime expression heatmap; D/ Pseudotime expression of CD63 in CP.

SF3B1 K700E Alters the Splicing of Metabolism and Stress Response Genes, Attenuating the Effect of DNMT3A Mutations in MDS

Myelodysplastic syndromes (MDS) are clonal stem cell disorders associated with low blood counts and a high risk of leukemic transformation. Genomic sequencing studies have demonstrated that MDS develops due to the acquisition of somatic mutations in hematopoietic stem cells (HSCs). Splicing factor 3b subunit 1 ( SF3B1) mutations, one of the most frequent mutations in MDS, are associated with lower-risk disease characterized by ineffective erythropoiesis. Our group and others have shown that mutations in SF3B1 cause aberrant mRNA splicing in human and murine HSCs, an expansion of long-term HSCs (LT-HSCs) in primary mice, but a competitive disadvantage in transplantation assays. DNMT3A, de novo methyltransferase 3A, is an epigenetic regulator mutated in ~10-15% of MDS. DNMT3A mutations are associated with unfavorable outcomes in MDS including a higher risk of leukemia transformation and shorter overall survival. Murine models of Dnmt3a mutations revealed that Dnmt3a-mutant HSCs have increased self-renewal over lineage-specific differentiation. SF3B1 and DNMT3A mutations co-occur more frequently than expected by chance and arise early in MDS pathogenesis, making them attractive therapeutic targets. MDS patients with SF3B1 and DNMT3A mutations have shorter progression-free survival than those with solely SF3B1 mutations. However, the mechanisms by which these mutations cooperate to cause HSC dysfunction and their possible combined role in leukemogenesis are not well understood. To investigate how Dnmt3a and Sf3b1 mutations disrupt HSC function in vivo we crossed two conditional knock-in mouse models of common point mutations in SF3B1 (K700E) and DNMT3A (R787H, murine equivalent to human DNMT3A R882H). We followed monthly blood counts and evaluated stem and progenitor cell composition in the bone marrow at 6 months. We found that Sf3b1/Dnmt3a double mutant mice display a similar phenotype to Sf3b1K700E mice, with a progressive anemia and an expansion in LT-HSCs. However, when assayed for HSC self-renewal potential, whole bone marrow and LT-HSCs isolated from Sf3b1/Dnmt3a mutant mice display increased serial colony formation capacities similar to Dnmt3a mutant hematopoietic stem and progenitor cells (HSPCs, 5 to 6 passages), but have a competitive disadvantage in secondary and tertiary transplant assays that is less severe than Sf3b1K700E HSPCs but distinct from the competitive advantage of Dnmt3a mutant HSPCs. Transcriptionally, double mutant HSCs clustered closer to Sf3b1 HSCs with shared expression of 74% of upregulated genes and 64% of downregulated genes and have decreased expression of stress response and metabolism genes. These results indicate Sf3b1 mutations may mitigate the preleukemic phenotype due to Dnmt3a mutations in MDS. To identify factors regulated by SF3B1 mutations that may mitigate the effect of DNMT3A loss on leukemic progression, we generated isogenic DNMT3A KO and SF3B1 K700E single and double mutant K562 cell lines using CRISPR/Cas9. Transcriptome analysis demonstrated that SF3B1K700E/DNMT3AKO double mutant clones clustered with and shared similar upregulated (69%) and downregulated (62%) genes as SF3B1 single mutant clones. Similar to our findings in murine HSPCs, pathway analysis of SF3B1 and SF3B1/DNMT3A mutant clones displayed enrichment of stress response and metabolic pathways. Alternative mRNA splicing analysis identified 13 events (3 alternative 3' splice site, 9 intron retention, and 1 skipped exon) in 8 genes that were upregulated in DNMT3A KO clones and downregulated in SF3B1 and SF3B1/DNMT3A mutant clones. We confirmed decreased protein expression of these mis-spliced genes in SF3B1 single and double mutant clones, but not DNMT3A KO clones. These genes include components of the integrated stress response pathway, which are also mis-spliced in SF3B1 and SF3B1/DNMT3A mutant MDS patient samples. Taken together our data suggest that aberrant splicing by mutant SF3B1 prevents metabolic reprogramming and decreases leukemic progression caused by DNMT3A mutation in MDS.

Acute Myeloid Leukemia (AML) from Older HSCs Is Resistant to Chemotherapy and Has an Altered DNA Damage Response

Acute myeloid leukemia (AML) is an aggressive malignancy that leads to the accumulation of immature myeloid precursors, resulting in progressive marrow failure and death. AML arises from the hematopoietic stem cell (HSC), either directly or through an early myeloid precursor. HSCs must survive for the entire human lifespan to provide the 200+ billion blood cells that are produced daily. They are subjected to the entirety of the aging process. Further, while AML is diagnosed in every age range, it disproportionately affects older patients, with a median age at diagnosis of 68. Older age, typically defined as 60 years of age or older, is a powerful, poor prognostic factor. Clinical trials show an estimated 5-year survival of less than 10% in these patients. Ten-year survival is even worse with one study estimating 2.4% of patients over 60 years of age alive at ten years when treated with chemotherapy. Importantly, this poor outcome is the result of disease that is significantly more resistant to therapy. Even though this clinical data is well known the underlying mechanisms of therapy resistance in older AML are unknown. To address this, we generated genetically identical AML cells from fetal, 12-week-old (Young) or 24-month-old (Old) murine HSCs. Retroviral vectors were used to express the oncogenic MLL-ENL fusion protein and NRas G12D. AML was confirmed by morphology, immunophenotype and all AML cells were lethal when injected into syngeneic recipients. Old AML cells demonstrated reduced autophagy, decreased NAD levels and impaired mitochondria consistent with a significant effect of biological aging. When treated with DNA damaging chemotherapy, Old AML cells were significantly more resistant than either Young or Fetal cells to both cytarabine and the anthracycline doxorubicin. To examine the DNA damage response AML cells were treated with doxorubicin and stained for the early DNA damage marker gH2AX. Old AML cells generated significantly more gH2AX than either Fetal or Young AML cells (Figure 1). We next examined the induction of p53 in response to treatment and in contrast to the gH2AX results Old AML cells induced the least amount of p53 in response to doxorubicin treatment (Figure 2). This data demonstrates that Old AML cells were more adept at recognizing DNA damage but least likely to induce p53 in response to it suggesting a difference in DNA repair. To better understand these differences RNA sequencing was performed and differentially expressed genes (DEGs) identified. When Fetal and Young AML cells were compared, no DEGs reached the false discovery threshold for significance, demonstrating that Fetal and Young AMLs have highly similar gene expression profiles. In contrast, when Old AML cells were compared to either Young or Fetal cells, hundreds of highly significant DEGs were identified, and KEGG pathway enrichment analysis revealed that several DNA repair pathways were significantly upregulated in Old AML. These include base excision repair (FDR p= 8.60E-06), mismatch repair (FDR p=3.26E-05), and nucleotide excision repair (FDR p= 0.00043). To further validate these pathways, the Beat AML TCGA dataset was assessed for the prognostic effect of increased expression of key genes in these pathways. Patients with increased expression of these genes had a trend toward worse survival (p=0.07) consistent with these pathways being clinically relevant. These data suggest that AML arising in older patients may overexpress DNA damage repair pathways leading to chemotherapy resistance and poor outcomes. Additional work on ways to increase sensitivity to Old AML cells is in progress.

The Sensitization of Murine Hematopoietic Progenitors and Stem Cells to DNA Damaging Agents By Quizartinib Is Associated with the Transcriptional Downregulation of DNA Damage Repair Genes

Background: The FLT3 inhibitor quizartinib induces quiescence of murine hematopoietic progenitors, and this affords their protection from S phase specific chemotherapies. In this study we investigated whether quizartinib can also protect hematopoietic progenitors from the cytotoxic effects of DNA damaging chemotherapies and radiation. Methods: C57BL/6 mice were primed with 30 mg/kg of quizartinib or vehicle (5% cyclodextrin) by oral gavage, and treated with DNA damaging chemotherapies, either cyclophosphamide, carboplatin, cisplatin, temozolomide or mitoxantrone 6 hours later, or 5.5Gy total body irradiation (TBI) after 9 hours. This TBI dose represents half the 11 Gy split dose used for bone marrow conditioning of C57BL/6 mice prior to HSC transplantation. Bone marrow cells were analyzed by flow cytometry after 3 to 6 days to identify progenitor and stem cell populations. Results: In contrast to quizartinib's protection against the cytotoxic effects of 5-FU and gemcitabine, all 5 DNA-damaging chemotherapies caused an increased loss of total bone marrow cellularity when mice were primed with quizartinib compared to vehicle primed controls. Flow cytometry analysis revealed that the numbers of myeloid and multipotent progenitors, and short-term and long-term hematopoietic stem cells (HSCs), were markedly lower in the quizartinib-treated mice. DNA damage and the resultant cell death caused by ionizing radiation is cell cycle specific, particularly for cells traversing from G 1 to S phases of the cell cycle. The ability of quizartinib to induce quiescence in myeloid and multipotent progenitors suggested that quizartinib may protect these cells from radiation. To test this, mice were dosed with vehicle or 30 mg/kg of quizartinib and 9 hours later exposed mice to 5.5 Gy of TBI. Analysis of bone marrow cells at day 6 revealed that total bone marrow cellularity and the numbers of progenitor and stem cells were markedly reduced in quizartinib-dosed mice compared to vehicle controls. For subsequent experiments mice were treated with 3 Gy TBI because the combination of quizartinib and 5.5 Gy caused severe myeloablation. As part of a series of experiments examining the kinetics of quizartinib's sensitization of bone marrow cells we tested whether the sensitization was maintained when mice were dosed with quizartinib 24 hours prior to 3 Gy TBI. Analysis of bone marrow cells 6 days later revealed that quizartinib dosed mice showed a significantly greater loss of total bone marrow cells, progenitor cells and stem cells compared to vehicle dosed mice (Figure 1). To identify possible mechanisms by which quizartinib sensitizes hematopoietic progenitors to DNA damaging agents we investigated quantitative gene expression profiling of the HPC7 hematopoietic progenitor cell line. These cells provide a suitable in vitro model since quizartinib induces their quiescence and provides protection against 5-FU. HPC7 cells were treated with 100 µM quizartinib, or vehicle, for 18 hr before RNA was harvested and sent for Next-generation sequencing. We found that quiescence induction in HPC7 cells by quizartinib correlated with the transcriptional downregulation of a wide range of genes involved in DNA damage repair pathways (including Brca1/2, Rad51, Palb2, Chek1 and Nbn), thus providing a possible mechanism for the enhanced cytotoxic effects of DNA damaging agents. Conclusions: Quizartinib sensitizes hematopoietic progenitors and stem cells to DNA-damaging agents, an effect that correlates with the transcriptional downregulation of many DNA damage repair genes. The enhanced depletion of hematopoietic progenitors and stem cells by DNA damaging agents in the presence of quizartinib has important clinical implications. Administering concurrent DNA damaging chemotherapy or radiotherapy with quizartinib treatment should be avoided. However, this interaction could facilitate a reduction in radiation/chemotherapy doses administered for bone marrow conditioning prior to HSC transplantation. By reducing these doses, but not compromising the conditioning process, non-hematopoietic side effects could be minimized and may allow currently excluded patients to be considered for HSC transplantation. Disclosure: This study was partly funded by Daiichi Sankyo.

TRIP13 Is a Fetal-Enriched Therapeutic Target in NUP98-JARID1A + Pediatric Non-Down Syndrome AMKL

Chromosomal rearrangements have been identified as the main drivers of pediatric Acute Megakaryoblastic Leukemia in absence of Down syndrome (non-DS-AMKL). The t(11;12) involving NUP98 and KDM5A/JARID1A (NJ) accounts for approximately 15% of pediatric non-DS-AMKL cases and correlates with poor prognosis. An important aspect of AMKL is the strong enrichment in pediatric patients with frequent occurrence in infants (disease within 2 years post birth). In this study, we aimed to explore the role of cellular ontogeny in NJ-driven non-DS-AMKL and highlight TRIP13 as a fetal-enriched NJ-specific vulnerability acting through P53 regulation. To investigate the mechanism of NJ transformation, we first transduced murine hematopoietic stem and progenitor cells (HSPCs) originating from fetal liver (mFL-HSPCs) and bone marrow of adult mice (mBM-HSPCs) with a lentiviral vector overexpressing the cDNA encoding human NJ. In line with other studies, NJacts as a potent oncogene in vitro, sustaining cell proliferation and arresting cell differentiation. Both mFL-HSPCs and mBM-HSPCs expressing NJ transformed and dominated the culture after 3-4 weeks with an immature immune-phenotype (Lin-, Sca1+ and c-Kit+). Notably, outgrowth of mFL-HSPCs was significantly accelerated compared to adult mBM-HSPCs, despite similar transduction rates. Furthermore, in vivo experiments revealed that NJ-expressing mFL-HSPCs displayed a more aggressive phenotype (latency of 36 days) compared to mBM-HSPCs cells expressing NJ (latency of 70 days; P Long-rank < 0.001). Thus, both our in vitro and in vivo experiments strongly indicate an impact of fetal cell background. Suspecting an impact of fetal gene programs on NJ-mediated transformation, we performed a CRISPR-Cas9 screening targeting fetal expression signatures with a library probing 880 genes found to be deregulated when comparing fetal versus adult murine and human primary HSPCs. By comparing our findings with two other fetal liver-derived leukemia models - representing DS AMKL and familial platelet disorder with predisposition to AML (FPD-AML) -, we identified TRIP13 as a high-confidence candidate gene with exclusive dependency in NJ-driven non-DS-AMKL. Comparing human AML cell lines and NJ-overexpressing human fetal liver cells (hFL-HSPCs) confirmed selective targeting of NJ-driven cells by TRIP13 loss. To explore the molecular mechanism of TRIP13 sensitivity, we performed RNAseq. Surprisingly, gene set expression analysis (GSEA) revealed one highly significantly enriched pathway, i.e. TP53 signaling. Rescue experiments of Trip13 knockout in NJ-driven non-DS-AMKL cells further supported this finding: the massive depletion upon loss of Trip13 was completely reverted by either re-expression of a human TRIP13 cDNA or by knockout of Trp53. Of note, neither activation of TP53 signaling nor cell depletion was seen in healthy hematopoietic cells from FL upon Trip13 perturbation. Finally, we aimed to leverage TRIP13 dependency therapeutically. To this end, we first explored Trip13-depletion in an in vivo setting. In a fluorescence-based competitive transplantation assay, Trip13-ablated leukemic blasts were significantly diminished in the bone marrow of recipient mice after 4 weeks, showing a mean depletion of 83%. Next, NJ-driven non-DS-AMKL mFL-HSPCs were treated with the TRIP13 inhibitor DCZ0415. Similarly to genetic TRIP13 ablation, DCZ0415-treated NJ mFL-HSPCs showed a significantly higher sensitivity to the drug compared to models of DS AMKL, FPD-AML, and healthy HSPCs with fold changes of 1.6 (p-value = 0.024), 2.3 (p-value = 0.001) and 2.0 (p-value = 0.008), respectively. To exploit our mechanistic knowledge on TRIP13 ablation-mediated P53 activation, we further combined DCZ0415 with the MDM2 inhibitor Idasanutlin. In line with our mechanistic data, a high synergy (Bliss synergy score = 9.7) of TRIP13 and MDM2 inhibition was observed. In conclusion, our study uncovers TRIP13 as a fetal-enriched vulnerability in NJ-driven non-DS-AMKL, mechanistically acting through P53, which we leveraged for a mechanism-driven treatment approach with dual TRIP13/MDM2 inhibition as a potential therapeutic strategy for the treatment of high-risk pediatric non-DS-AMKL.

Targeting IGF2BP3 Enhances Anti-Leukemic Effects of Menin-MLL Inhibition in MLL-AF4 Leukemia

Background: MLL-rearranged leukemias are a clinically challenging and biologically unique subtype of leukemias associated with poor prognosis. While novel therapeutic strategies have been primarily directed at epigenetic dysregulation, post-transcriptional gene regulatory mechanisms have emerged as important mediators in leukemogenesis and have the unexplored potential to be potent combinatorial therapeutic targets. Previously, we found that the RNA-binding protein IGF2BP3 is a critical regulator of MLL-AF4 leukemogenesis and represents a promising therapeutic target. Methods: We studied the combined effects of targeting IGF2BP3 and the Menin-MLL interaction in MLL-AF4 driven leukemia in vitro and in vivo, using genetic inhibition through CRISPR-Cas9 mediated deletion of Igf2bp3 and pharmacologic inhibition of the Menin-MLL interaction with commercially available inhibitors MI-503, MI-463, and MI-538. In vitro, we tested the human B-cell acute lymphoblastic leukemia cell lines, SEM, RS4;11 and NALM6, and MLL-Af4-transformed murine hematopoietic stem and progenitor cells (herein referred to as MLL-Af4 Lin-), derived from bone marrow of Cas9 mice. Results: Depletion of Igf2bp3 sensitized MLL-AF4 leukemia to the negative effects of Menin-MLL inhibition on leukemic cell growth, colony formation and leukemic initiating cells in vitro. Mechanistically, we found that both Igf2bp3 depletion and Menin-MLL inhibition led to increased differentiation in vitro and in vivo in functional readouts and by gene expression analyses. Both MI-503 treatment and IGF2BP3 knockdown in MLL-Af4 Lin- cells showed a shift towards more differentiated colony morphologies in colony formation assays, decreased expression of c-Kit and increased expression of maturation markers by flow cytometry, and morphologic changes consistent with increased differentiation. To gain insight into these phenotypic findings, we examined gene expression from MLL-Af4 Lin- cells with IGF2BP3 knockdown and treated with MI-503. Metascape analysis revealed significant enrichment in pathways involved in cell differentiation and activation, particularly in leukocytes. This was observed in both I3KO and MI-503 treated cells, with significant overlap in shared differentially expressed genes in both conditions. Next, we looked at the overlap between differentially expressed genes with MI-503 treatment and IGF2BP3 knockdown with targets identified from MLL-AF4 ChIP and IGF2BP3 CLIP in CD11b+ and MLL-Af4 Lin- cells. We found significant overlap between differentially expressed genes with MI-503 treatment and IGF2BP3 CLIP targets, suggesting that IGF2BP3 directly regulates genes that are affected by Menin-MLL inhibition. Furthermore, we saw overlap between differentially expressed genes with IGF2BP3 knockdown and MLL-AF4 ChIP targets identified in SEM and RS4;11 cells. These patterns highlight the interaction between the MLL-AF4 and IGF2BP3 transcriptomes and suggest a mechanism for the synergistic inhibition of leukemia by targeting both MLL-AF4 and IGF2BP3. To examine the combined effects of IGF2BP3 knockdown and Menin-MLL inhibition on leukemic engraftment and survival in vivo, we transplanted MLL-Af4 Lin- cells, depleted (or non-depleted) for IGF2BP3 and treated with MI-503 at 0.5 μM for 5 days, to initiate leukemia. We observed a significant decrease in peripheral blood leukemic engraftment with MI-503 treatment and IGF2BP3 knockdown. IGF2BP3 knockdown had a greater effect on survival and attenuating disease than MI-503 alone and showed enhanced anti-leukemic effects in combination. Conclusions: Our work shows that IGF2BP3 is an oncogenic amplifier of MLL-AF4 mediated leukemogenesis and is a potent therapeutic target and suggests a novel combinatorial approach to targeting leukemia at both the transcriptional and post-transcriptional level.