Survival Of Hematopoietic Progenitor Cells Research Articles

Novel Small Molecule, UTS-1401, as a Radioprotector for Total-Body Irradiation.

We report on a new radioprotector, UTS-1401, a small molecule that was synthesized (by one of us, JS) and evaluated here for its radioprotective effect against total-body irradiation (TBI). Female and male NIH Swiss mice were subjected to TBI at doses of 6.5, 7.5 and 8.5 Gy either with or without a 24 h pretreatment of UTS-1401 given ip and observed for 30 days. Survival rates were significantly increased when mice were treated with UTS-1401 compared to those not treated. The radioprotective effect of UTS-1401 was drug-dose dependent for male mice exposed to 8.5 Gy TBI with 150 mg/kg of UTS-1401 as the optimal dose. The radioprotective effect of UTS-1401 on female mice exposed to 8.5 Gy TBI was observed at 50, 100, and 150 mg/kg, with no dose response relationship noted. Female mice were more radioresistant than male mice with LD50/30 values of 7.8 Gy vs. 6.8 Gy, respectively. Weight changes after UTS-1401 alone showed a significant body weight increase at 150 mg/kg. Both the ip and iv route for UTS-1401 were similarly effective for male mice exposed to 8 Gy TBI. Further analysis using an endogenous spleen colony assay demonstrated that pretreatment of UTS-1401 for up to 72h prior to TBI protected both spleen weight and hematopoietic stem cells with a treated/untreated ratio between 2.0 and 3.2 for the latter for times between 0.5 h and 72 h. A separate invivo study showed that pretreatment of UTS-1401 protected bone marrow CFU-GM for mice exposed to TBI. In summary, UTS-1401 is a promising small-molecule radioprotective agent as demonstrated by whole animal, hematopoietic stem cell and bone marrow myeloid progenitor cell survival.

Not just for lymphoid cells: The role of the noncanonical NF-κB signaling pathway in early and late myelopoiesis with a focus on hypereosinophilic disorders.

The noncanonical NF-κB pathway is involved in lymphoid organ development, B cell maturation, and cytokine production. However, new research has demonstrated that this pathway is also key for the orderly and sequential maturation of myeloid cells, including neutrophils and eosinophils. When this pathway is disrupted or constitutively activated, aberrations in hematopoietic stem and progenitor cell (HSPC) survival and proliferation, as well as subsequent granulopoiesis and eosinophilopoiesis are affected. Disturbance of such a coordinated and delicate process can manifest in devastating clinical disease including acute and chronic myeloid leukemias (AML and CML, respectively), pre-leukemic processes such as myelodysplastic syndrome (MDS) or hyperinflammatory conditions like Hypereosinophilic Syndrome (HES). In this review, we will discuss the molecular machinery within the noncanonical NF-κB pathway, crosstalk with the canonical NF-κB pathway, murine models of noncanonical signaling, as well as how aberrations in this pathway manifest in leukemic or hyperinflammatory disease with a focus on HES. Potential and promising drug therapies will also be discussed, emphasizing the noncanonical NF-κB pathway as a potential target for improved treatment for patients suffering from leukemia or idiopathic HES. The hope is that review of such mechanisms and treatments may eventually result in findings that aid physicians in rapidly diagnosing and more accurately classifying patients suffering from such complex and overlapping hematopoietic diseases.

Abstract LB268: Discovery of a potent, selective and brain penetrating PARP1 inhibitor, XZP-7797

Abstract First-generation PARP1/2 inhibitors are approved for the treatment of cancers, such as ovarian, prostate, pancreas, and breast cancers, with maximal activity against tumors harboring BRCA1/2 mutation or homologous recombination deficiency (HRD). However, PARP1/2 inhibitors in clinical use have severe hematological toxicities due to the inhibition of PARP2, which has been shown to play an important role in the survival of hematopoietic progenitor cells. Furthermore, the current data indicate that synthetic lethality with BRCA mutations is primarily caused by PARP1 inhibition and trapping, whereas PARP2 inhibition and trapping are not critical for anticancer activity. Therefore, the development of selective PARP1 inhibitors is expected to reduce the hematological adverse effects associated with PARP2 inhibition while maintaining the required efficacy. XZP-7797 is a potent, selective, and brain-penetrating PARP1 inhibitor with trapping ability. In biochemical assays, XZP-7797 is more than 1000-fold selective for PARP1 over PARP2 and other members of the PARP family, including PARP3, PARP5a, PARP5b, and PARP6. XZP-7797 can significantly inhibit MDA-MB-436 cell proliferation in vitro (IC50=2.4nM). In MDA-MB-436 tumor cells, XZP-7797 inhibits PARylation and increases γH2AX level in a concentration-dependent manner. XZP-7797 demonstrates rapid tumor regression in a dose-dependent manner in the BRCA1 mutant MDA-MB-436 breast cancer xenograft model. In a pancreatic cancer xenograft model, XZP-7797 can also significantly inhibit tumor growth. To evaluate the ability of XZP-7797 to cross the blood-brain barrier, an intracranial xenograft model of breast cancer is used. The results show that XZP-7797 can significantly inhibit the growth of intracranial tumors in mice at a dose of 10 mg/kg, demonstrating the brain-penetrating ability of XZP-7797. Additionally, XZP-7797 has great PK profiles in preclinical species after oral dosing and good ADME properties. In a 14-day toxicology study of XZP-7797 in SD Rats, XZP-7797 has no hematological adverse effects at the exposure that is 10 times higher than the exposure required to achieve superior efficacy. Citation Format: Yeqing Gao, Yuqian Lu, Bo Chen, Benke Jiang, Feng Wang, Genyan Zhang, Xifeng Ma, Dapeng Zhou, Chunmin Shi, Yunling Wang, Zhipeng Liu, Jun Sun, Huimin Zhou, Mei Xu, Jiakui Li, Bin Liu. Discovery of a potent, selective and brain penetrating PARP1 inhibitor, XZP-7797 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB268.

Abstract 4525: Discovery of a highly potent and selective PARP1 inhibitor with superior PK and safety profiles

Abstract PARP inhibitors have been an effective treatment option for cancers deficient in homologous recombination repair, such as ovarian, breast, prostate, and pancreatic cancers, particularly those harboring BRCA mutations. However, their severe overlapping hematological and gastrointestinal toxicities have limited their clinical use, especially in the combination therapies with other anti-cancer agents. More recently, studies have shown that inhibiting PARP1 mainly drives anti-tumor activity while inhibiting PARP2 contributes to the hematological toxicity such as anemia due to its essential role in the survival of hematopoietic stem and progenitor cells. Thus, there is an urgent need for developing a selective PARP1 inhibitor. In this study, we report the discovery and characterization of a unique PARP1 inhibitor, ACE-86225106. This molecule is highly selective at inhibiting PARP1 over PARP2 as demonstrated in biochemical (72x) and DNA-trapping (131x) assays. It demonstrates potent anti-proliferative activities against BRCA mutant cancer cell lines (IC50 = 1-9 nM) but not against BRCA wild type cancer cells (IC50 >30,000 nM). As compared to the BRCA mutant cancer cells, this molecule is much less active at inhibiting the differentiation of CD34+ hematopoietic stem cells (IC50 ≈ 450-1,400 nM), i.e.: erythroid, myeloid and megakaryocyte. The results of these studies show that ACE-86225106 is a highly potent and selective PARP1 inhibitor. This molecule has a unique PK profile characterized by its long half-life and low volume of distribution. As such, it demonstrates robust efficacy at low dose (1 mg/kg) with a less frequent dosing schedule (once a week). Importantly, there was no significant body weight loss observed in the treatment group even at much higher doses (10 mg/kg), but there was a significant body weight gain. The GLP toxicology studies have confirmed its superior safety profile as the maximum recommended safe starting first-in-human dose was >5-fold higher than the predicted human efficacious dose. Taken together, ACE-86225106 is a highly potent, selective PARP1 inhibitor with excellent PK and safety profile and expected to have a better safety profile, deeper and more durable clinical responses, and broader utility in the clinic as a preferred combination partner with other anti-cancer therapies. A phase 1 clinical trial to evaluate its safety, PK, and clinical utility is to begin in early 2024. Citation Format: Yi Wei, Wenming Li, Zehui Li, Manhua Li, Kai Kang, Zhiming Wen, Tinggui Yin, Kuo-Long Yu, Genshi Zhao, Hongbin Yuan. Discovery of a highly potent and selective PARP1 inhibitor with superior PK and safety profiles [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 4525.

Polycom Group Protein Mel18 Inhibits Hematopoietic Stem Cell Self-Renewal through Repressing the Expression of Genes Important for Cellular Senescence and Proliferation

Polycomb group (PcG) proteins are epigenetic gene silencers that have been implicated in stem cell maintenance and cancer development. Genetic and biochemical studies indicate that Polycomb group proteins exist in at least two protein complexes, Polycomb repressive complex 2 (PRC2) and Polycomb repressive complex 1 (PRC1), that act in concert to initiate and maintain stable gene repression. Bmi1 and Mel18 are two major homologs of the PCGF subunit within the canonical PRC1 complex. While Bmi1 is a positive regulator of hematopoietic stem cell (HSC) and leukemia stem cell (LSC) self-renewal, the role of Mel18 in normal and malignant hematopoiesis is not fully understood. To further determine the role of Mel18 in hematopoiesis, we have generated Mel18 conditional knockout mice ( Mel18 f/f-Mx1Cre +). Acute deletion of Mel18 in the hematopoietic compartment did not affect the frequency and survival of hematopoietic stem and progenitor cells (HSPCs). To determine the impact of Mel18 deficiency on HSPC proliferation, we performed serial replating assays and found that loss of Mel18 increases the replating potential of HSPCs in vitro. To determine the role of Mel18 in HSC self-renewal, we first performed serial bone marrow (BM) transplantation assays and found that Mel18 null BM cells show increased engraftment in both primary and secondary transplantation assays. Limiting dilution transplantation showed that there were more functional HSCs in the BM of Mel18 knockout mice. We then performed HSC transplantation assays and found that loss Mel18 enhances HSC self-renewal. Thus, we demonstrated that Mel18 inhibits HSC self-renewal and decreases the number of functional HSCs in vivo. To understand the mechanism by which Mel18 inhibits HSC self-renewal and proliferation, we performed RNA-seq using Mel18 +/+ and Mel18 -/- HSCs. We then performed gene set enrichment analysis (GSEA) to identify potential Mel18 target genes important for HSC behavior. We found that HSC gene signatures are enriched in Mel18 -/- HSCs. We also found that leukemia stem cell gene signatures and CBF-mutant-AML genes are significantly enriched in Mel18 -/- HSCs, suggesting that loss of Mel18 may prime normal HSCs for leukemic transformation. Notably, our RNA-seq assay showed that senescence-related genes, including S100a9 and S100a8, were down-regulated in Mel18 -/- HSPCs. Importantly, loss of Mel18 significantly decreased the number of senescent HSPCs. To understand how Mel18 regulates gene transcription, we performed ATAC-seq assays in Mel18 +/+ and Mel18 -/- HSPCs. Mel18 null HSPCs showed decreased chromatin accessibility near both S100a8 and S100a9 genes. We confirmed that both S100a8 and S100a9 are downregulated in Mel18 -/- HSPCs compared to Mel18 +/+ HSPCs. Given that cell cycle arrest is a common feature of cellular senescence, we also examined the expression of cell cycle related genes in Mel18 -/- HSPCs. The expression of cell cycle regulators, including CDK4 and CCND2, is increased in Mel18 -/- HSPCs. Mel18 can bind to a specific DNA sequence, 5‘-GACTNGACT-3‘, thereby repressing the expression of target genes. Mel18 directly binds to the CCND2 gene locus as revealed by ChIP-seq assays in embryonic stem cells, suggesting that Mel18 may directly repress CCND2 expression in HSPCs. In summary, we demonstrate that Mel18 inhibits HSC self-renewal via repressing the expression of genes that are important for cellular senescence and proliferation.

GATA1 and GATA2 Sense/Antisense Transcriptional Switching in Early Hematopoietic Development

The GATA1 and GATA2 genes play essential roles in hematopoietic development. GATA1 is the master regulator of erythropoiesis and GATA2 is required for the expansion and survival of hematopoietic progenitor and stem cells, as well as the development of mast cells. A switch in GATA expression occurs during erythropoiesis, with silencing of the GATA2 gene and increased expression of GATA1. A GATA2 antisense transcript ( GATA2-AS1) has been associated with silencing of GATA2 expression. We have identified a novel GATA1 antisense promoter located in the first intron that opposes the GATA1 promoter and generates a spliced antisense transcript complementary to the SUV39H1 H3K9 methyltransferase gene. The competition between opposing sense and antisense promotersin the GATA1 and GATA2 genes may play a central role in the switching of GATA expression that occurs during hematopoiesis. To address this hypothesis, gene expression analysis of cells expressing sense versus antisense transcripts from the GATA1 or GATA2 genes was performed using 5'scRNAseq data from in vitro differentiated CD34 cells. Cells expressing only sense or only antisense GATA1 transcriptswere readily detected and comprised the majority (90%) of cells with GATA1 transcripts. Differential gene expression analysis of cells with GATA1 sense-only versus cells with GATA1 antisense-only showed association of sense transcription with erythrocyte genes, whereas antisense expression was associated with a mast cell phenotype and upregulation of GATA2 transcript levels. Conversely, GATA2 antisense expressing cells had increased GATA1 transcription and an erythrocyte phenotype, and GATA2 sense expressing cells demonstrated mast cell-specific gene expression. Single cell RNAseq analysis of HL60 cells induced to differentiate in vitro identified significant changes in GATA2 gene expression. Induction of granulocyte differentiation in HL60 cells treated with DMSO resulted in increased GATA2 antisense transcription, whereas PMA induction of monocyte differentiation was associated with increased GATA2 sense transcription. Taken together these results indicate a central role for sense/antisense promoter switching in hematopoiesis.

MiR-223-3p promotes genomic stability of hematopoietic progenitors after radiation

When hematopoietic cells are overwhelmed with ionizing radiation (IR) DNA damage, the alternative non-homologous end-joining (aNHEJ) repair pathway is activated to repair stressed replication forks. While aNHEJ can rescue cells overwhelmed with DNA damage, it can also mediate chromosomal deletions and fusions, which can cause mis-segregation in mitosis and resultant aneuploidy. We previously reported that a hematopoietic microRNA, miR-223-3p, normally represses aNHEJ. We found that miR-223-/- mice have increased survival of hematopoietic stem and progenitor cells (HSPCs) after sublethal IR. However, this came at the cost of significantly more genomic aberrancies, with miR-223-/- hematopoietic progenitors having increased metaphase aberrancies, including chromothripsis, and increased sequence abnormalities, especially deletions, which is consistent with aNHEJ. These data imply that when an HSPC is faced with substantial DNA damage, it may trade genomic damage for its own survival by choosing the aNHEJ repair pathway, and this choice is regulated in part by miR-223-3p.

Advances in Understanding the Links between Metabolism and Autophagy in Acute Myeloid Leukemia: From Biology to Therapeutic Targeting.

Autophagy is a highly conserved cellular degradation process that regulates cellular metabolism and homeostasis under normal and pathophysiological conditions. Autophagy and metabolism are linked in the hematopoietic system, playing a fundamental role in the self-renewal, survival, and differentiation of hematopoietic stem and progenitor cells, and in cell death, particularly affecting the cellular fate of the hematopoietic stem cell pool. In leukemia, autophagy sustains leukemic cell growth, contributes to survival of leukemic stem cells and chemotherapy resistance. The high frequency of disease relapse caused by relapse-initiating leukemic cells resistant to therapy occurs in acute myeloid leukemia (AML), and depends on the AML subtypes and treatments used. Targeting autophagy may represent a promising strategy to overcome therapeutic resistance in AML, for which prognosis remains poor. In this review, we illustrate the role of autophagy and the impact of its deregulation on the metabolism of normal and leukemic hematopoietic cells. We report updates on the contribution of autophagy to AML development and relapse, and the latest evidence indicating autophagy-related genes as potential prognostic predictors and drivers of AML. We review the recent advances in autophagy manipulation, combined with various anti-leukemia therapies, for an effective autophagy-targeted therapy for AML.

Transient Monosomy 7 Is a Rare Event in Young Children with SAMD9L Syndrome

Monosomy 7 and deletion 7q are among the most common chromosomal aberrations in pediatric myeloid malignancies and generally associated with an increased risk of rapid progression to advanced neoplasia, warranting allogeneic hematopoietic stem cell transplantation (HSCT). However, several cases of transient monosomy 7 and spontaneous hematologic recovery upon loss of monosomy 7 in young children have been described in the literature. In several cases, germline mutations in sterile alpha-motif domain-containing protein 9 (SAMD9) and SAMD9-like (SAMD9L), both located on chromosome 7q, have been identified. Gain of function (GOF) mutations in SAMD9 and SAMD9L cause phenotypically overlapping syndromes that predispose to pediatric myelodysplastic syndrome (MDS) and are associated with non-hematological anomalies and variable immunodeficiency. GOF SAMD9/9L mutations inhibit cell proliferation and reduce cell survival of hematopoietic stem and progenitor cells (HSPCs). This may exert strong selection pressure on HSPCs to acquire somatic aberrations that improve their competitive fitness. To no surprise, a distinctive feature of SAMD9/9L syndromes is their propensity for somatic genetic reversion, including somatic loss of function mutations in SAMD9/9L in cis, uniparental isodisomy 7q, and non-random loss of chromosome 7 or 7q of the mutant allele. Multiple hematopoietic subclones with clone-defining somatic mutations can exist in the bone marrow and their composition may change over time due to the exceptionally high plasticity of hematopoiesis in young children. Consequently, monosomy 7 may spontaneously disappear in young patients with SAMD9/9L syndromes. Earlier observations had suggested that somatic compensatory mutations may be more frequent in SAMD9L than in SAMD9 mutated patients. Here, we investigate the natural history of young children with SAMD9L syndrome and monosomy 7. We report on seven patients with SAMD9L syndrome and monosomy 7 presenting before the age of 5 years in whom an initial surveillance strategy was electively chosen rather than timely allogeneic HSCT. All patients presented with refractory cytopenia of childhood with monosomy 7 at a median age of 1.0 years (range: 0.4-3.1 years). Six patients had an additional non-hematologic phenotype, including prematurity (P2, P5), small for gestational age (P1, P2, P7), cerebellar atrophy and developmental delay (P1), bilateral cleft lip and palate (P2), macrocephaly (P3, P6), shorter thumbs (P6), and big eye bulbs and dystrophy (P7). Immunodeficiency was present in five patients ranging from hypogammaglobulinemia (P1, P2, P5, P6, P7) to B and NK cell deficiency patients (P2, P5, P6, P7). At a median follow-up of 35 months (range: 10-51 months), allogeneic HSCT had been employed in four patients for severe neutropenia and/or bacterial infection (P1, P7), persistence of monosomy 7 beyond the age of 5 years (P5) or disease progression to MDS with excess blasts (P2). One patient (P2) suffered transplant-related mortality, while the remaining patients were alive at last follow-up. Three patients did not require definitive treatment. Two of the three patients had complete loss of the monosomy 7 clone with concomitant recovery of blood counts (P4, P6), a third patient had normalized blood counts, but cytogenetic and molecular studies at follow-up were missing (P3). Secondary somatic mutations were detected in six patients. Five patients carried somatic SAMD9L mutations in cis (P2, P3, P4, P6, P7). Two patients with spontaneous loss of monosomy 7 carried large somatic SAMD9L subclones that were stable over time and presumably maintained hematopoiesis (P4, P6). In addition, two patients carried somatic mutations in oncogenic driver genes (P2, P5). P2, who developed MDS-EB 14 months after diagnosis, had two RUNX1 mutations at the time of progression. P5 had one RUNX1 and one EZH2 mutation, but without evidence of disease progression. Our results suggest that loss of monosomy 7 can occur in young patients with SAMD9L syndrome but is a rare event. If a watch and wait strategy is chosen, close monitoring with frequent bone marrow examinations including SAMD9L sequencing, cytogenetics and search for somatic oncogenic events is advised. Importantly, presence of severe infections, severe neutropenia, increase in blast percentage, and persistence of monosomy 7 beyond the age of 5 years should prompt timely allogeneic HSCT.

Granulocyte colony-stimulating factor has a sex-dependent positive effect in the maternal immune activation-induced autism model.

The medical intervention for autism spectrum disorder (ASD) is restricted to ameliorating comorbid situations. Granulocyte colony-stimulating factor (G-CSF) is a growth factor that enhances the proliferation, differentiation, and survival of hematopoietic progenitor cells. In the present study, we aimed to investigate the effects of G-CSF in a maternal immune activation-induced autism model. Sixteen female and six male Wistar adult rats were included in the study. After 21 days, 48 littermates (eight male controls, eight female controls, 16 male lipopolysaccharide [LPS]-exposed rats, and 16 female LPS-exposed rats) were divided into groups. Sixteen male LPS-exposed and 16 female LPS-exposed rats were divided into saline and G-CSF treatment groups. In male rats, the LPS-exposed group was found to have significantly higher levels of TNF-α, IL-2, and IL-17 than the LPS-exposed G-CSF group. Levels of nerve growth factor, brain PSD-95, and brain GAD67 were higher in the LPS-exposed G-CSF group than in the LPS-exposed group in male rats. In female rats, brain NGF levels were similar between groups. There was no difference between groups in terms of brain GAD 67 levels. Brain PSD-95 levels were higher in the control group than in both the LPS-exposed and LPS-exposed G-CSF groups in female rats. Both neuronal CA1 and neuronal CA2 levels were lower, and the GFAP immunostaining index (CA1) and GFAP immunostaining index (CA3) were higher in the LPS-exposed group than in the LPS-exposed G-CSF group in male rats. However, neuronal count CA1 and neuronal count CA3 values were found to be similar between groups in female rats. The present research is the first to demonstrate the beneficial effects of G-CSF on core symptoms of ASD experimentally depending on male sex. G-CSF can be a good candidate for ameliorating the core symptoms of ASD without serious side effects in males.

Abstract 2609: AZD9574 is a novel, brain penetrant PARP-1 selective inhibitor with activity in an orthotopic, intracranial xenograft model with aberrant DNA repair

Abstract The Poly (ADP-ribose) polymerase (PARP) family has numerous essential functions in cellular processes such as transcription, chromatin remodelling, DNA damage response and repair as well as apoptosis. PARP inhibition blocks base excision repair and results in conversion of SSBs to DNA double-strand break (DSBs). DSBs are the most deleterious form of DNA damage that can be generated by exogenous DNA damaging agents or endogenous replication stress. DSBs can be repaired by homologous recombination repair (HRR) or non-homologous end joining (NHEJ). The physiological importance of HRR is underscored by the observation of genomic instability in HRR-deficient (HRD+) cells and, importantly, the association of cancer predisposition and developmental defects with mutations in HRR genes. PARP1 and PARP2 are required for SSB repair, while PARP1 is also involved in the repair of DNA double-strand breaks (DSBs) and replication fork damage. Recent reports suggest the PARP1 inhibition is sufficient to elicit an anti-proliferative effect and that PARP2 is essential for the survival of hematopoietic stem and progenitor cells in animal models.AZD9574 is a novel brain penetrant PARP1 inhibitor that acts by selectively inhibiting and trapping PARP1 at the sites of SSBs. AZD9574 exhibited >8000-fold selectivity for PARP1 compared to PARP2 and other members of the PARP family (PARP2, PARP3, PARP5a and PARP6) in biochemical assays. While AZD9574 inhibited PARP1 enzymatic activity in all tested cell lines irrespective of the HRR status (IC50 range between 0.3 - 2 nM), colony formation assay in isogenic cell lines pairs confirmed higher potency and selectivity towards HRD+ models (DLD1 BRCA2-/-; SKOV-3 BRCA2-/- and SKOV-3 PALB2-/-). For example, AZD9574 IC50 in the BRCA2-/- DLD1 cells was 1.38 nM compared to IC50 > 40 µM BRCA2wt cells, which corresponds to a ~20,000-fold greater efficacy in the BRCA2-/-cells (ratio of AZD9574 IC50 in BRCA2wt divided by BRCA2-/-) compared to the wild type parental line. In vivo, AZD9574 demonstrated dose-dependent efficacy in a BRCA1 mutant MDA-MB-436 subcutaneous xenograft model. Anti-tumour effects of AZD9574 were manifested by pronounced growth regressions that were durable after treatment withdrawal. An intracranial xenograft model of breast cancer brain metastases was developed to assess the efficacy of AZD9574 in the context of blood-brain barrier penetrance. Treatment of animals with established intracranial lesions using a dose of 3 mg/kg AZD9574 showed sustained tumour growth suppression resulting in a significantly extended survival of tumour-bearing mice. Collectively, we believe that our data support the development of AZD9574 as a potential therapy for patients with HRD+ breast cancer whose disease has spread to the brain. Citation Format: Kunzah Jamal, Anna Staniszewska, Jacob Gordon, Shenghua Wen, Frank McGrath, Gregory Dowdell, Dominic Kabbabe, Giuditta illuzzi, Matthew Griffin, Barry R. Davies, Petra Hamerlik. AZD9574 is a novel, brain penetrant PARP-1 selective inhibitor with activity in an orthotopic, intracranial xenograft model with aberrant DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2609.

GATA2 Deficiency in Adult Life Is Characterized by Phenotypic Diversity and Delayed Diagnosis.

The transcription factor GATA2 plays a key role in the survival and self-renewal of hematopoietic stem and progenitor cells. Autosomal dominant variants in GATA2 cause a broad spectrum of heterogeneous phenotypes. Here, we present our experience with GATA2 deficiency in a retrospective multicenter analysis of computerized medical records of adult patients (age ≥18 years) treated between 2018 and 2022 at Shaare Zedek Medical Center in Jerusalem and Sheba Tel-Hashomer Medical Center in Ramat Gan, Israel. Two male and two female patients with GATA2 deficiency were identified. Three of the patients presented with symptoms in adult life and all patients were diagnosed as adults. Age at presentation was 10.5-36 years and age at diagnosis 24-47 years. Diagnosis was delayed in all patients by 1-24.5 years. The phenotypic diversity was notable. Patients presented with myelodysplastic syndrome (n=2), pulmonary alveolar proteinosis (n=1), and recurrent viral (n=1), bacterial (n=3), and mycobacterial (n=1) infections. Bone marrow biopsy revealed cytogenetic abnormalities in one patient (monosomy 7). Patients were diagnosed by exome sequencing (n=3) and Sanger sequencing of the coding exons in GATA2 (n=1). Novel heterozygous GATA2 variants (c.177C>A, p.Y59* and c.610dup, p.R204Pfs*78) were identified in two patients. Immune workup revealed B cell lymphopenia and monocytopenia in all tested patients. One patient died from overwhelming sepsis despite all patients being treated with antibiotics and anti-mycobacterials. Our cohort highlights the phenotypic diversity, late presentation, and delayed diagnosis of GATA2 deficiency. Increased awareness of this primary immune deficiency presenting in adult life is needed and should involve a high index of suspicion.

DDX41 is needed for pre- and postnatal hematopoietic stem cell differentiation in mice.

SummaryDDX41 is a tumor suppressor frequently mutated in human myeloid neoplasms, but whether it affects hematopoiesis is unknown. Using a knockout mouse, we demonstrate that DDX41 is required for mouse hematopoietic stem and progenitor cell (HSPC) survival and differentiation, particularly of myeloid lineage cells. Transplantation of Ddx41 knockout fetal liver and adult bone marrow (BM) cells was unable to rescue mice from lethal irradiation, and knockout stem cells were also defective in colony formation assays. RNA-seq analysis of Lin−/cKit+/Sca1+Ddx41 knockout cells from fetal liver demonstrated that the expression of many genes associated with hematopoietic differentiation were altered. Furthermore, differential splicing of genes involved in key biological processes was observed. Our data reveal a critical role for DDX41 in HSPC differentiation and myeloid progenitor development, likely through regulating gene expression programs and splicing.

Hemgn Protects Hematopoietic Stem and Progenitor Cells Against Transplantation Stress Through Negatively Regulating IFN‐γ Signaling (Adv. Sci. 5/2022)

Hematopoietic Stem and Progenitor Cells In article number 2103838, Xiao-Ming Yang, Chang-Yan Li and co-workers identify a critical regulator-HEMGN, that is required for hematopoietic stem and progenitor cells (HSPCs) homing, survival, and reconstitution of blood cells during bone marrow transplantation (BMT). HEMGN acts as a protector which limits interferon-γ (IFN-γ) signaling by suppressing nuclear Stat1 tyrosine phosphorylation and assists HSPCs to combat the transplantation stress. Targeted Hemgn may be used to improve conditioning regimens and engraftment during bone marrow transplantation.

Leukemia Inhibitory Factor Promotes Survival of Hematopoietic Progenitors Ex Vivo and Is Post-Translationally Regulated by DPP4.

Hematopoietic cells are regulated in part by extracellular cues from cytokines. Leukemia inhibitory factor (LIF) promotes survival, self-renewal, and pluripotency of mouse embryonic stem cells (mESC). While genetic deletion of LIF affects hematopoietic progenitor cells (HPCs), the direct effect of LIF protein exposure on HPC survival is not known. Furthermore, post-translational modifications (PTM) of LIF and their effects on its function have not been evaluated. We demonstrate that treatment with recombinant LIF preserves mouse and human HPC numbers in stressed conditions when growth factor addition is delayed ex vivo. We show that Lif is upregulated in response to irradiation-induced stress. We reveal novel PTM of LIF where it is cleaved twice by dipeptidyl peptidase 4 (DPP4) protease so that it loses its 4 N-terminal amino acids. This truncation of LIF down-modulates LIF's ability to preserve functional HPC numbers ex vivo following delayed growth factor addition. DPP4-truncated LIF blocks the ability of full-length LIF to preserve functional HPC numbers. This LIF role and its novel regulation by DPP4 have important implications for normal and stress hematopoiesis, as well as for other cellular contexts in which LIF and DPP4 are implicated.

Extracellular DEK Treatment Mimics Hypoxic Blockade of Extra Physiologic Oxygen Stress in Human and Mouse Hematopoietic Cells

Hematopoietic stem cell (HSC) and progenitor cell (HPC) self-renewal, proliferation, survival, and function are regulated by extracellular signals from cytokines and chemokines. DEK, a nuclear regulator of chromatin availability, was recently shown to also function as an extracellular protein that regulates HSC and HPC by inducing signaling through the CXC chemokine receptor 2 (CXCR2), enhancing pools of long-term stem cells while decreasing pools of functional HPC [Capitano et al., JCI, 2019, 129(6):2555-70]. Due to differing effects of extracellular (ec)DEK treatment on HSC growth and function compared to the effects on HPC, we hypothesized that DEK differentially regulates HSC compared to HPC. To explore this, we pulse-treated purified human cord blood (CB) CD34+ cells for 18 hours with recombinant human ecDEK protein. We then isolated by flow cytometry stringently immunophenotypically-defined human HSC, multipotent progenitors (MPP), common myeloid progenitors (CMP), and granulocyte-macrophage progenitors (GMP) and performed mRNA-sequencing to profile ecDEK-dependent transcriptomes of these separate populations of HSC/HPC. Interestingly, there are widely different ranges of effects on the transcriptomes of the different isolated HSC/HPC populations. CMP had the most differentially expressed (DE) genes induced by DEK treatment, followed by HSC and MPP, and GMP had relatively few changes, suggesting DEK treatment may primarily affect HSC and earlier HPC. HSC exhibited the strongest magnitude of changes in DE genes. Fast gene set enrichment analysis (FGSEA) revealed that GMP showed upregulation of gene programs associated with leukocyte migration and downregulation of gene programs regulated by MYC, CMP showed upregulation of gene programs associated with myeloid cell development and downregulation of lysine methyltransferase activity, and MPP and HSC both upregulated cytokine signaling responses in response to DEK treatment, though HSC also exhibited a downregulation of genes associated with cell cycle. Despite the identified unique changes upon DEK treatment in these different HSC/HPC populations, we were most interested to discover that all examined cell types demonstrated upregulation of genes associated with hypoxic responses in cells, and a concordant downregulation of genes that are frequently downregulated by hypoxic exposure following DEK treatment. This suggested to us that DEK may stimulate overlapping pathways related to hypoxia responses and/or response to exposure to extra physiologic oxygen. In fact, the effects on HSC and HPC induced by ecDEK is similar with previous work demonstrating that isolating HSC/HPC at 3% O 2 compared to ambient air conditions (~21% O 2) leads to a preserved pool of functional HSC but a lower number of functional HPC, due to a phenomenon termed extracellular physiologic shock/stress (EPHOSS) [Mantel et al., Cell, 2015, 161(7):1553-65]. To explore whether DEK regulates similar stress response pathways that are associated with EPHOSS, we assessed the effects of ecDEK treatment on HSC/HPC isolated at 3% O 2. Mice were treated with DEK in vivo, then bone marrow cells were isolated at 3% O 2 in a hypoxia chamber. Cells were kept at 3% O 2 or split to ambient air to allow equilibration to a higher oxygen tension and then assessed for HSC and functional HPC numbers. In contrast to untreated HSC/HPC isolated at 3% O 2, ecDEK treated cells isolated at 3% O 2 did not exhibit further increases in LT-HSC numbers or decreases in pools of functional HPC over ecDEK treatment alone identified by colony assays. Isolation at 3% O 2 followed by in vitro treatment with DEK showed similar results. This suggests ecDEK may neutralize the effects of EPHOSS by acting on similar gene programs that are induced by ambient air exposure. We also demonstrate that ecDEK prevents the ex vivo apoptosis of CB HSC/HPC populations, again suggesting that ecDEK is preventing the cellular stress associated with ambient air exposure and ex vivo growth, resulting in a preservation of HSC. Taken together, these data show that ecDEK acts as an extracellular signaling protein that prevents cellular stress by activating gene programs that overlap with hypoxia associated gene programs. This has important implications for the role of ecDEK in normal and diseased hematopoiesis, as well as clinical implications wherein ecDEK may be used to mimic the HSC preserving effects of isolation at 3% O 2. DisclosuresMarkovitz: University of Michigan: Patents & Royalties: H84T BanLec and of the H84T-driven CAR construct.

Editorial introductions.

Editorial introductions.

Heterodimerization of TPO and IFNγ Impairs Human Hematopoietic Stem/Progenitor Cell Signaling and Survival in Chronic Inflammation

Thrombopoietin (TPO) is the main regulator of hematopoietic stem and progenitor cell (HSPC) self-renewal and survival. Upon binding to its receptor, c-MPL, TPO activates cell signaling through JAK-STAT and other pathways. In recent clinical trials, eltrombopag, a small molecule c-MPL agonist, improved trilineage hematopoiesis in subjects with severe aplastic anemia (SAA). Paradoxically, TPO levels are already markedly elevated in these patients. To explain this paradox, we previously showed that IFNγ, a key proinflammatory cytokine implicated in the destruction of HSPCs in SAA, inhibited TPO signaling in human CD34+ HSPCs cultured in the presence of both cytokines. In contrast, eltrombopag could evade this inhibition in vitro, resulting in improved maintenance of progenitors in clonogenic CFU assays, and long-term repopulating cells in NSG transplantation models compared with TPO-containing cultures (Cheng et al., ASH abstract 2016). In this study, we sought to characterize the mechanisms by which eltrombopag evades IFNγ blockade of c-MPL signaling. Because activation of both c-MPL and IFNγ receptor by their respective ligands induces negative regulatory feedback mechanisms from the SOCS family, we first measured expression of SOCS proteins in CD34+ cells exposed to IFNγ and TPO or eltrombopag. SOCS expression was equally upregulated by TPO and eltrombopag in the presence of IFNγ, suggesting an alternative explanation for eltrombopag's ability to escape IFNγ-induced perturbation of TPO signaling. Because TPO and eltrombopag distinctively bind to the extracellular or juxtamembrane domain of c-MPL, respectively, we hypothesized that IFNγ may decrease binding affinity of TPO, but not eltrombopag, to c-MPL. We used microscale thermophoresis (MST) to assess the impact of IFNγ on TPO binding to its receptor. As previously reported, we showed that TPO and c-MPL interact via both a high affinity (KD, app<0.11 ± 0.04 nM) and a low affinity (KD, app=1100 ± 130 nM) binding site. In contrast, IFNγ had no interaction with c-MPL. Remarkably, addition of 100-molar excess IFNγ prevented binding of TPO to c-MPL at the low affinity site, but had no impact on the high affinity binding site (KD, app<0.20 ± 0.04 nM) (Panel A). To explain this result, we hypothesized that TPO and IFNγ may directly interact and form heteromeric complexes that hinder binding to c-MPL, whereas the non-peptide small molecule eltrombopag could conceivably evade that process. Strikingly, MST assays revealed a specific, one-site heterodimeric interaction between TPO and IFNγ (KD, app=540 ± 30 nM), while no heterodimer formation was observed between either TPO or IFNγ and other early-acting cytokines (SCF and Flt3L) (Panel B). These data suggest that TPO:IFNγ heterodimers may be responsible for the hindered TPO:c-MPL low-affinity interaction observed in the presence of IFNγ. To confirm this finding, we investigated whether TPO-induced c-MPL dimerization was interrupted in the presence of IFNγ. Using raster image correlation spectroscopy (RICS) to determine average diffusion coefficients of GFP-tagged c-MPL in live cells, we have preliminarily found that IFNγ negatively affects c-MPL dimerization in cells cultured with TPO, but not with eltrombopag. Further studies are underway. Taken together, our data provide a new and provocative paradigm to explain the observed negative impact of the proinflammatory cytokine IFNγ on human HSPC maintenance in bone marrow failure syndromes, and the ability of a small molecule (eltrombopag) to evade this inhibition. We propose that, under chronic inflammatory conditions, IFNγ specifically heterodimerizes with TPO, resulting in (i) occlusion of the low-affinity binding site of TPO to c-MPL, (ii) impaired receptor dimerization, (iii) perturbation of TPO-induced signaling pathways, and (iv) decreased survival of human HSPCs (Panel C). This new understanding could have far-reaching clinical implications for other disorders of chronic inflammation. [Display omitted] DisclosuresHuntsman:Novartis: Research Funding. Cheng:Novartis: Research Funding. Larochelle:Novartis: Research Funding; Novartis: Research Funding; StemCell Technologies: Patents & Royalties: StemDiff Hematopoietic Kit.

Nfix  Promotes Survival of Immature Hematopoietic Cells Via Regulation of C-Mpl

Hematopoietic stem and progenitor cells (HSPC) are necessary for life-long blood production and replenishment of the hematopoietic system during stress. We recently identified the transcription factor nuclear factor I/X (Nfix) as a critical regulator of hematopoietic stem and progenitor cell (HSPC) survival post-transplantation. Nfix -depleted HSPC are lost from the marrow between 7-10 days post-transplant and residual Nfix -depleted HSPC display elevated levels of apoptosis at these time-points. To further characterize the anti-apoptotic role of Nfix in HSPCs, we overexpressed Nfix in Lineage- Sca-1+ c-Kit+ (LSK) cells that were then maintained ex vivo under serum-free conditions with supportive cytokines and monitored the cultures for cell surface phenotype, growth, vector selection, and apoptosis. Ectopic expression of Nfix in primary mouse HSPCs had no significant effect on the expression of lineage markers compared to controls, but did appear to accelerate the loss of immunophenotypic LSK cells, as well as colony forming units (p = 0.023). Nfix -overexpressing HSPCs were extended in their ex vivo culture lifespan from about 20 days to 40 days, and were steadily selected for in culture. HSPCs overexpressing Nfix also displayed hypersensitivity to supportive cytokines and reduced apoptosis when subjected to cytokine deprivation compared to controls (p = 0.032). NFIX has been shown to potentially influence transcription of several known regulators of HSC biology. Here, we show that ectopic Nfix resulted in elevated levels of c-Mpl transcripts and cell surface protein on primary murine HSPCs as well as increased phosphorylation of STAT5, which is known to be activated down-stream of c-MPL. Consequently, Bcl-XL, an anti-apoptotic factor induced by STAT5, was also significantly upregulated in Nfix -overexpressing cells by two weeks of culture compared to controls (p = 0.0038). Blocking c-MPL signaling by removal of thrombopoietin or addition of a c-MPL neutralizing antibody significantly negated the anti-apoptotic effect of Nfix overexpression on cultured HSPCs (p = 0.045, p = 0.0098, respectively). Further, NFIX was capable of transcriptional activation of a proximal c-Mpl promoter fragment (p = 0.0056). In sum, these data suggest that NFIX-mediated up-regulation of c-Mpl transcription can protect primitive hematopoietic cells from stress ex vivo . DisclosuresNo relevant conflicts of interest to declare.

The AF9-Binding Domain of DOT1L Is Critical for Its Recruitment By MLL Fusion Proteins in Leukemia and Contributes to Its Functions in Normal Hematopoiesis

Mixed lineage leukemia (MLL) is a very aggressive therapy resistant leukemia. MLL-AF9 is one of the most common MLL-fusion proteins that recruits disruptor of telomeric silencing 1-like (DOT1L), to MLL-target genes HOXA9/MEIS1. DOT1L is a histone methyltransferase that catalyzes methylation of H3K79, a gene activating mark, that is essential for leukemogenesis and normal hematopoiesis. Our lab and others mapped the 10 amino acid (865 - 874) binding site on DOT1L, which binds to the ANC1 homology domain (AHD) domain of AF9. We also identified a point mutation, I867A, sufficient to disrupt the AF9-DOT1L interaction in vitro. We demonstrated that DOT1L lacking the 10 amino acids (Δ10) were unable to support transformation by ML-AF9. We hypothesize that by disrupting the AF9-DOT1L protein-protein interaction (PPI), we will be able to inhibit leukemogenesis and potentially circumvent the negative consequences of DOT1L loss in normal bone marrow. We used a genetic approach to explore the role of DOT1L recruitment in leukemogenesis and normal hematopoiesis.We generated cell lines derived from MLL-AF9-driven DOT1Lf/f CreER(T2)+ bone marrow by transducing DOT1L constructs, including wild type (WT-DOT1L), deletion of 10 amino acids (Δ10-DOT1L), point mutant (I867A-DOT1L), and an enzymatically inactive mutant (RCR-DOT1L). Both Δ10 and I867A mutants inhibited leukemogenesis similarly to the RCR mutant. This inhibited proliferation was coupled with a decrease in H3K79me2, a decrease in HOXA9/Meis1 gene expression, cellular differentiation, and induction of apoptosis demonstrating that disruption of the AF9-DOT1L PPI mimics the phenotype of as the enzymatic inhibition, deeming it as a potential therapeutic target for this cancer.To explore the impact of disrupting the AF9-DOT1L interaction in normal bone marrow, we transduced Mx-Cre+ DOT1Lf/f bone marrow with either WT-DOT1L, Δ10-DOT1L, or RCR-DOT1L constructs and injected these cells into lethally irradiated B6-CD45.1 recipients. After hematopoietic reconstitution, recipients were treated with poly(I:C) to inactivate endogenous DOT1L. In the absence of DOT1L's methyltransferase activity (RCR-DOT1L), transduced hematopoietic progenitors underwent rapid failure. Interestingly, expression of the Δ10-DOT1L mutant was sufficient to support short-term hematopoiesis but insufficient to sustain long-term hematopoietic stem cells (LT-HSCs). Together these results show that blocking DOT1L's recruitment eliminates the transformative potential of the MLL-AF9 oncogene and that while DOT1L's function is critical for all hematopoietic progenitor cell survival, removing the DOT1L AF9 binding domain allows for short term hematopoiesis.To further explore the role of AF9 role in recruiting DOT1L in MLL-AF9 leukemia and normal hematopoiesis, using high throughput screening approach, we identified small molecules that bind to the AF9 AHD domain. These compounds inhibit proliferation of cells harboring the MLL-AF9 fusion, decrease H3K79me2, and induce apoptosis similar to Δ10-DOT1L and I867A-DOT1L mutants. We are currently working to further characterize their on-target activity in cells, and test their effects on normal bone marrow, with the ultimate goal to validate additional avenues for therapeutic intervention for MLL-rearranged leukemia. DisclosuresNo relevant conflicts of interest to declare.