Blood Progenitor Research Articles

Radiation Therapy can be Safely Incorporated Into Pre-Transplant Treatment Regimens for Patients With Multiple Myeloma

<h3>Purpose/Objective(s)</h3> Primary treatment of multiple myeloma (MM) often involves systemic induction therapy (SIT) followed by autologous stem cell transplant (ASCT). Radiation therapy (RT) is sometimes used for palliation; however, many practitioners avoid RT out of concern that future peripheral blood progenitor cell (PBPC) collection required for ASCT may be compromised. We retrospectively examined the possible effect of RT on PBPC collection. <h3>Materials/Methods</h3> We reviewed the charts of 732 MM patients (pts) seen from 1997–2016 at our institution and included pts who received RT prior to PBPC collection for planned ASCT. RT plans (both MM and non-MM RT) were reviewed to estimate the percentage (%) bone marrow (BM) treated using published estimates of skeletal BM distribution. Statistics were performed using Pearson correlations and t-tests. <h3>Results</h3> Of 732 MM pts, ASCT was planned for 485 pts; of these, 223 pts received RT prior to PBPC collection and were included in the final cohort. 133 pts (60%) were male. Median age at PBPC collection was 59 years (range 33—80). For SIT, pts received combination regimens including the following agents: bortezomib (142 pts, 64%); lenalidomide (111 pts, 50%); alkylators (46 pts, 21%). Nine pts (4%) received dexamethasone alone. RT plans and/or a description of the fields, were available to estimate treated BM% for 221 pts. The median cumulative BM% treated per pt was 6.7 (range 0.0—47.4). The median RT dose was 24 Gy (range 10.0—75.6 Gy). Mobilization was performed using granulocyte-colony stimulating factor (G-CSF) alone (189 pts, 85%), G-CSF with plerixafor (15 pts, 7%), or chemotherapy (19 pts, 8%). PBPC collection data was available for 199 pts. A median of 7.8 × 10⁶ CD34⁺/kg PBPCs (range 0.5—54.8) were collected in a median of 3 (1—9) apheresis procedures. 196 pts (99%) collected more than 2.0 × 10⁶ CD34⁺/kg (minimum PBPC no. required for engraftment), and 167 pts (84%) collected more than 5.0 × 10⁶ CD34⁺/kg (preferred no. of PBPCs collected). The number of PBPCs collected was not associated with BM% treated (<i>P</i> = 0.15) or RT dose (<i>P</i> = 0.56). The number of apheresis procedures performed was not associated with BM% treated (<i>P</i> = 0.54) or RT dose (<i>P</i> = 0.85). The number of PBPCs collected did not differ significantly for pts receiving RT to the pelvis (<i>P</i> = 0.96), lumbar spine (<i>P</i> = 0.35), or thoracic spine (<i>P</i> = 0.059). Time to platelet engraftment was longer for patients with higher %BM treated (<i>P</i> = 0.02). Eleven pts did not undergo a confirmed ASCT due to: pt preference (3 pts), trial therapy (1 pt), comorbidities (1 pt), election for hospice (1 pt), inadequate collection (4 pts) and inadequate follow up (1 pt). <h3>Conclusion</h3> Among pts with MM, RT prior to ASCT did not prevent adequate PBPC collection or impair successful ASCT. RT must be carefully planned and delivered to ensure safe incorporation into pre-ASCT treatment regimens.

KAT2A complexes ATAC and SAGA play unique roles in cell maintenance and identity in hematopoiesis and leukemia

AbstractEpigenetic histone modifiers are key regulators of cell fate decisions in normal and malignant hematopoiesis. Their enzymatic activities are of particular significance as putative therapeutic targets in leukemia. In contrast, less is known about the contextual role in which those enzymatic activities are exercised and specifically how different macromolecular complexes configure the same enzymatic activity with distinct molecular and cellular consequences. We focus on KAT2A, a lysine acetyltransferase responsible for histone H3 lysine 9 acetylation, which we recently identified as a dependence in acute myeloid leukemia stem cells and that participates in 2 distinct macromolecular complexes: Ada two-A-containing (ATAC) and Spt-Ada-Gcn5-Acetyltransferase (SAGA). Through analysis of human cord blood hematopoietic stem cells and progenitors, and of myeloid leukemia cells, we identify unique respective contributions of the ATAC complex to regulation of biosynthetic activity in undifferentiated self-renewing cells and of the SAGA complex to stabilization or correct progression of cell type–specific programs with putative preservation of cell identity. Cell type and stage-specific dependencies on ATAC and SAGA-regulated programs explain multilevel KAT2A requirements in leukemia and in erythroid lineage specification and development. Importantly, they set a paradigm against which lineage specification and identity can be explored across developmental stem cell systems.

Label-free separation of neuroblastoma patient-derived xenograft (PDX) cells from hematopoietic progenitor cell products by acoustophoresis

BackgroundGraft-contaminating tumor cells correlate with inferior outcome in high-risk neuroblastoma patients undergoing hematopoietic stem cell transplantation and can contribute to relapse. Motivated by the potential therapeutic benefit of tumor cell removal as well as the high prognostic and diagnostic value of isolated circulating tumor cells from stem cell grafts, we established a label-free acoustophoresis-based microfluidic technology for neuroblastoma enrichment and removal from peripheral blood progenitor cell (PBPC) products.MethodsNeuroblastoma patient-derived xenograft (PDX) cells were spiked into PBPC apheresis samples as a clinically relevant model system. Cells were separated by ultrasound in an acoustophoresis microchip and analyzed for recovery, purity and function using flow cytometry, quantitative real-time PCR and cell culture.ResultsPDX cells and PBPCs showed distinct size distributions, which is an important parameter for efficient acoustic separation. Acoustic cell separation did not affect neuroblastoma cell growth. Acoustophoresis allowed to effectively separate PDX cells from spiked PBPC products. When PBPCs were spiked with 10% neuroblastoma cells, recoveries of up to 98% were achieved for PDX cells while more than 90% of CD34+ stem and progenitor cells were retained in the graft. At clinically relevant tumor cell contamination rates (0.1 and 0.01% PDX cells in PBPCs), neuroblastoma cells were depleted by more than 2-log as indicated by RT-PCR analysis of PHOX2B, TH and DDC genes, while > 85% of CD34+ cells could be retained in the graft.ConclusionThese results demonstrate the potential use of label-free acoustophoresis for PBPC processing and its potential to develop label-free, non-contact tumor cell enrichment and purging procedures for future clinical use.

Demonstration and implications of IL-3 upregulation of CD25 expression on human mast cells

CD25+ human mast cells (huMCs) have been reported in patients with monoclonal mast cell diseases and in rare association with inflammation. However, the regulation of CD25 expression on huMCs and the possible biologic consequences remain poorly understood. We sought to identify conditions that would upregulate CD25 expression on huMCs and to explore possible functional implications. huMCs were cultured from peripheral blood progenitor cells over 6 to 8 weeks. Expression of CD25 was determined by fluorescence-activated cell sorting and soluble CD25 by ELISA. Signal transducer and activator of transcription 5 (STAT5) phosphorylation induced by IL-2 in huMCs, regulatory T (Treg) cells, or in cocultured huMCs and Treg cells was examined by fluorescence-activated cell sorting. Addition of IL-3 to CD34+ progenitors at the initiation of huMC cultures in the presence of stem cell factor and IL-6 upregulated the expression of CD25 in developing huMCs and resulted in shedding of soluble CD25 into the media. Removal of IL-3 after the first week of culture did not affect subsequent expression of CD25. Furthermore, addition of IL-3 14 days after the initiation of the culture did not induce significant CD25 expression. Treatment with anti-IL-3 antibody or the Janus kinase inhibitor tofacitinib blocked IL-3-induced CD25 upregulation. Binding of IL-2 to CD25+ huMCs did not induce STAT5 phosphorylation. However, coincubation of Treg cells with CD25+ huMCs pretreated with IL-2 was sufficient to result in STAT5 phosphorylation in Treg cells. IL-3 promotes CD25 expression and shedding by huMCs. Although CD25+ huMCs do not respond to IL-2, they bind IL-2 and may act as a reservoir of IL-2 to then activate lymphocytes.

A gap-junction-mediated, calcium-signaling network controls blood progenitor fate decisions in hematopoiesis

Stem cell homeostasis requires coordinated fate decisions among stem cells that are often widely distributed within a tissue at varying distances from their stem cell niche. This requires a mechanism to ensure robust fate decisions within a population of stem cells. Here, we show that, in the Drosophila hematopoietic organ, the lymph gland (LG), gap junctions form a network that coordinates fate decisions between blood progenitors. Using live imaging of calcium signaling in intact LGs, we find that blood progenitors are connected through a signaling network. Blocking gap junction function disrupts this network, alters the pattern of encoded calcium signals, and leads to loss of progenitors and precocious blood cell differentiation. Ectopic and uniform activation of the calcium-signaling mediator CaMKII restores progenitor homeostasis when gap junctions are disrupted. Overall, these data show that gap junctions equilibrate cell signals between blood progenitors to coordinate fate decisions and maintain hematopoietic homeostasis.

Dual targeting of JAK2 and ERK interferes with the myeloproliferative neoplasm clone and enhances therapeutic efficacy

Myeloproliferative neoplasms (MPN) show dysregulated JAK2 signaling. JAK2 inhibitors provide clinical benefits, but compensatory activation of MAPK pathway signaling impedes efficacy. We hypothesized that dual targeting of JAK2 and ERK1/2 could enhance clone control and therapeutic efficacy. We employed genetic and pharmacologic targeting of ERK1/2 in Jak2V617F MPN mice, cells and patient clinical isolates. Competitive transplantations of Jak2V617F vs. wild-type bone marrow (BM) showed that ERK1/2 deficiency in hematopoiesis mitigated MPN features and reduced the Jak2V617F clone in blood and hematopoietic progenitor compartments. ERK1/2 ablation combined with JAK2 inhibition suppressed MAPK transcriptional programs, normalized cytoses and promoted clone control suggesting dual JAK2/ERK1/2 targeting as enhanced corrective approach. Combined pharmacologic JAK2/ERK1/2 inhibition with ruxolitinib and ERK inhibitors reduced proliferation of Jak2V617F cells and corrected erythrocytosis and splenomegaly of Jak2V617F MPN mice. Longer-term treatment was able to induce clone reductions. BM fibrosis was significantly decreased in MPLW515L-driven MPN to an extent not seen with JAK2 inhibitor monotherapy. Colony formation from JAK2V617F patients’ CD34+ blood and BM was dose-dependently inhibited by combined JAK2/ERK1/2 inhibition in PV, ET, and MF subsets. Overall, we observed that dual targeting of JAK2 and ERK1/2 was able to enhance therapeutic efficacy suggesting a novel treatment approach for MPN.

Definitive hematopoietic stem cells minimally contribute to embryonic hematopoiesis.

SUMMARYHematopoietic stem cells (HSCs) are rare cells that arise in the embryo and sustain adult hematopoiesis. Although the functional potential of nascent HSCs is detectable by transplantation, their native contribution during development is unknown, in part due to the overlapping genesis and marker gene expression with other embryonic blood progenitors. Using single-cell transcriptomics, we define gene signatures that distinguish nascent HSCs from embryonic blood progenitors. Applying a lineage-tracing approach to selectively track HSC output in situ, we find significantly delayed lymphomyeloid contribution. An inducible HSC injury model demonstrates a negligible impact on larval lymphomyelopoiesis following HSC depletion. HSCs are not merely dormant at this developmental stage, as they showed robust regeneration after injury. Combined, our findings illuminate that nascent HSCs self-renew but display differentiation latency, while HSC-independent embryonic progenitors sustain developmental hematopoiesis. Understanding these differences could improve de novo generation and expansion of functional HSCs.

Alginate-Chitosan Microencapsulated Cells for Improving CD34+ Progenitor Maintenance and Expansion

Protocols for isolation, characterization, and transplantation of hematopoietic stem cells (HSCs) have been well established. However, difficulty in finding human leucocyte antigens (HLA)-matched donors and scarcity of HSCs are still the major obstacles of allogeneic transplantation. In this study, we developed a double-layered microcapsule to deliver paracrine factors from non-matched or low-matched HSCs to other cells. The umbilical cord blood-derived hematopoietic progenitor cells, identified as CD34+ cells, were entrapped in alginate polymer and further protected by chitosan coating. The microcapsules showed no toxicity for surrounding CD34+ cells. When CD34+ cells-loaded microcapsules were co-cultured with bare CD34+ cells that have been collected from unrelated donors, the microcapsules affected surrounding cells and increased the percentage of CD34+ cell population. This study is the first to report the potency of alginate-chitosan microcapsules containing non-HLA-matched cells for improving proliferation and progenitor maintenance of CD34+ cells.

NOTCH-mediated exvivo expansion of human hematopoietic stem and progenitor cells by culture under hypoxia.

SummaryActivation of NOTCH signaling in human hematopoietic stem/progenitor cells (HSPCs) by treatment with an engineered Delta-like ligand (DELTA1ext-IgG [DXI]) has enabled ex vivo expansion of short-term HSPCs, but the effect on long-term repopulating hematopoietic stem cells (LTR-HSCs) remains uncertain. Here, we demonstrate that ex vivo culture of human adult HSPCs with DXI under low oxygen tension limits ER stress in LTR-HSCs and lineage-committed progenitors compared with normoxic cultures. A distinct HSC gene signature was upregulated in cells cultured with DXI in hypoxia and, after 21 days of culture, the frequency of LTR-HSCs increased 4.9-fold relative to uncultured cells and 4.2-fold compared with the normoxia + DXI group. NOTCH and hypoxia pathways intersected to maintain undifferentiated phenotypes in cultured HSPCs. Our work underscores the importance of mitigating ER stress perturbations to preserve functional LTR-HSCs in extended cultures and offers a clinically feasible platform for the expansion of human HSPCs.

Activation and Contraction of Human "Vascular" Smooth Muscle Cells Grown From Circulating Blood Progenitors.

Blood outgrowth smooth muscle cells (BO-SMCs) offer the means to study vascular cells without the requirement for surgery providing opportunities for drug discovery, tissue engineering, and personalized medicine. However, little is known about these cells which meant that their therapeutic potential remains unexplored. Our objective was to investigate for the first time the ability of BO-SMCs and vessel-derived smooth muscle cells to sense the thromboxane mimetic U46619 by measuring intracellular calcium elevation and contraction. U46619 (10–6 M) increased cytosolic calcium in BO-SMCs and vascular smooth muscle cells (VSMCs) but not in fibroblasts. Increased calcium signal peaked between 10 and 20 s after U46619 in both smooth muscle cell types. Importantly, U46619 (10–9 to 10–6 M) induced concentration-dependent contractions of both BO-SMCs and VSMCs but not in fibroblasts. In summary, we show that functional responses of BO-SMCs are in line with VSMCs providing critical evidence of their application in biomedical research.

Drosophila as a Model to Study Cellular Communication Between the Hematopoietic Niche and Blood Progenitors Under Homeostatic Conditions and in Response to an Immune Stress.

In adult mammals, blood cells are formed from hematopoietic stem progenitor cells, which are controlled by a complex cellular microenvironment called “niche”. Drosophila melanogaster is a powerful model organism to decipher the mechanisms controlling hematopoiesis, due both to its limited number of blood cell lineages and to the conservation of genes and signaling pathways throughout bilaterian evolution. Insect blood cells or hemocytes are similar to the mammalian myeloid lineage that ensures innate immunity functions. Like in vertebrates, two waves of hematopoiesis occur in Drosophila. The first wave takes place during embryogenesis. The second wave occurs at larval stages, where two distinct hematopoietic sites are identified: subcuticular hematopoietic pockets and a specialized hematopoietic organ called the lymph gland. In both sites, hematopoiesis is regulated by distinct niches. In hematopoietic pockets, sensory neurons of the peripheral nervous system provide a microenvironment that promotes embryonic hemocyte expansion and differentiation. In the lymph gland blood cells are produced from hematopoietic progenitors. A small cluster of cells called Posterior Signaling Centre (PSC) and the vascular system, along which the lymph gland develops, act collectively as a niche, under homeostatic conditions, to control the balance between maintenance and differentiation of lymph gland progenitors. In response to an immune stress such as wasp parasitism, lymph gland hematopoiesis is drastically modified and shifts towards emergency hematopoiesis, leading to increased progenitor proliferation and their differentiation into lamellocyte, a specific blood cell type which will neutralize the parasite. The PSC is essential to control this emergency response. In this review, we summarize Drosophila cellular and molecular mechanisms involved in the communication between the niche and hematopoietic progenitors, both under homeostatic and stress conditions. Finally, we discuss similarities between mechanisms by which niches regulate hematopoietic stem/progenitor cells in Drosophila and mammals.

Ultrasound-assisted continuous-flow synthesis of PEGylated MIL-101(Cr) nanoparticles for hematopoietic radioprotection

Metal-organic frameworks (MOFs) are useful as drug delivery carriers with high loading capacity and excellent biocompatibility. We fabricated a new drug carrier based on MIL-101(Cr) environmentally and loaded it with 47.2 wt% WR-1065 (active metabolite of amifostine). Moreover, the permeability and stability of these nanoparticles increased after PEGylation by the N-hydroxysuccinimide active ester protocol. Then, a “green” continuous-flow system equipped with an ultrasound applicator was newly designed to prepare the nanoparticles under the effect of acoustic cavitation. Response surface methodology (RSM) was used to optimize the large-scale process conditions with Box-Behnken design to obtain high space-time yield (5785 kg m−3 day−1). These less toxic MOFs nanoparticles increased cell viability by scavenging the accumulated reactive oxygen species and resisting DNA damage after irradiation. They are capable of mitigating radiation injury, achieving a 30-d survival rate of 90% in mice after lethal total body irradiation (8.0 Gy). This countermeasure significantly improved the peripheral blood cell count, hematopoietic stem and progenitor cells frequency, and clonogenic function of hematopoietic progenitor cells. It probably prevents irradiation-induced hematopoietic damage through the p53-dependent apoptotic pathway. Therefore, ultrasound-assisted continuous-flow synthesis is a sustainable method to produce MOFs on a large scale for radioprotection.

Relish plays a dynamic role in the niche to modulate Drosophila blood progenitor homeostasis in development and infection.

Immune challenges demand the gearing up of basal hematopoiesis to combat infection. Little is known about how during development, this switch is achieved to take care of the insult. Here, we show that the hematopoietic niche of the larval lymph gland of Drosophila senses immune challenge and reacts to it quickly through the nuclear factor-κB (NF-κB), Relish, a component of the immune deficiency (Imd) pathway. During development, Relish is triggered by ecdysone signaling in the hematopoietic niche to maintain the blood progenitors. Loss of Relish causes an alteration in the cytoskeletal architecture of the niche cells in a Jun Kinase-dependent manner, resulting in the trapping of Hh implicated in progenitor maintenance. Notably, during infection, downregulation of Relish in the niche tilts the maintenance program toward precocious differentiation, thereby bolstering the cellular arm of the immune response.

A Conserved Role for Asrij/OCIAD1 in Progenitor Differentiation and Lineage Specification Through Functional Interaction With the Regulators of Mitochondrial Dynamics.

Mitochondria are highly dynamic organelles whose activity is an important determinant of blood stem and progenitor cell state. Mitochondrial morphology is maintained by continuous fission and fusion and affects stem cell proliferation, differentiation, and aging. However, the mechanism by which mitochondrial morphology and dynamics regulate cell differentiation and lineage choice remains incompletely understood. Asrij/OCIAD1 is a conserved protein that governs mitochondrial morphology, energy metabolism and human embryonic stem cell (hESC) differentiation. To investigate the in vivo relevance of these properties, we compared hESC phenotypes with those of Drosophila hematopoiesis, where Asrij is shown to regulate blood progenitor maintenance by conserved mechanisms. In concordance with hESC studies, we found that Drosophila Asrij also localizes to mitochondria of larval blood cells and its depletion from progenitors results in elongated mitochondria. Live imaging of asrij knockdown hemocytes and of OCIAD1 knockout hESCs showed reduced mitochondrial dynamics. Since key regulators of mitochondrial dynamics actively regulate mitochondrial morphology, we hypothesized that mitochondrial fission and fusion may control progenitor maintenance or differentiation in an Asrij-dependent manner. Knockdown of the fission regulator Drp1 in Drosophila lymph gland progenitors specifically suppressed crystal cell differentiation whereas depletion of the fusion regulator Marf (Drosophila Mitofusin) increased the same with concomitant upregulation of Notch signaling. These phenotypes were stronger in anterior progenitors and were exacerbated by Asrij depletion. Asrij is known to suppress Notch signaling and crystal cell differentiation. Our analysis reveals that synergistic interactions of Asrij with Drp1 and Marf have distinct impacts on lymph gland progenitor mitochondrial dynamics and crystal cell differentiation. Taken together, using invertebrate and mammalian model systems we demonstrate a conserved role for Asrij/OCIAD1 in linking mitochondrial dynamics and progenitor differentiation. Our study sets the stage for deciphering how regulators of mitochondrial dynamics may contribute to functional heterogeneity and lineage choice in vertebrate blood progenitors.

Mutation Signatures of Pediatric Acute Myeloid Leukemia and Normal Blood Progenitors Associated with Differential Patient Outcomes

Acquisition of oncogenic mutations with age is believed to be rate limiting for carcinogenesis. However, the incidence of leukemia in children is higher than in young adults. Here we compare somatic mutations across pediatric acute myeloid leukemia (pAML) patient-matched leukemic blasts and hematopoietic stem and progenitor cells (HSPCs), as well as HSPCs from age-matched healthy donors. HSPCs in the leukemic bone marrow have limited genetic relatedness and share few somatic mutations with the cell-of-origin of the malignant blasts, suggesting polyclonal hematopoiesis in pAML patients. Compared to normal HSPCs, a subset of pAML cases harbored more somatic mutations and a distinct composition of mutational process signatures. We hypothesize these cases might have arisen from a more committed progenitor. This subset had better outcomes than pAML cases with mutation burden comparable to age-matched healthy HSPCs. Our study provides insights into the etiology and patient stratification of pAML.

Abstract 1265: Discovery of GT19077, a c-Myc/Max protein-protein Interaction (PPI) small molecule inhibitor, and GT19506 a c-Myc PROTAC molecule, for targeting c-Myc-driven blood cancers and small cell lung cancers

Abstract c-Myc is an oncogenic transcriptional factor, which form a dimer with Max to activate its transcription activity, driving tumor initiation, progression and poor prognosis in 80% of all tumor types. IGH/Myc genomic translocations have been identified in B-cell lymphoma (15-100%). The amplification of Myc family members, MYC, MYCN, or MYCL have been revealed in 20% patients with small cell lung cancers (SCLC). Therefore It is highly warranted to discover and develop novel c-Myc inhibitors targeting c-Myc dependent tumors (Lymphoma and SCLC) with acceptable therapeutic index. Here we described the discovery of GT19077, a c-Myc/Max PPI small molecule inhibitor, and GT19506, a c-Myc PROTAC, for targeting Myc-dependent tumors. GT-19077 was demonstrated to disrupt c-Myc/Max dimer with an IC50 of 1.0 µM in PPI assays and degraded c-Myc protein with IC50 of 0.34 µM in HL-60 cells measured in c-Myc ELISA assays, which was confirmed by Western Blotting (WB). GT19506 (PROTAC) effectively degraded c-Myc proteins in HL-60 cells with an IC50 of 0.10 µM. The selectivity of both compounds against the physiologic function of c-Myc was determined in growth factor-stimulated hematopoietic progenitor cells by using c-Myc ELISA assays. GT19077 and GT19506 were less potent at degrading c-Myc in growth-factor driven hematopoietic cells with IC50 of 2.36 and 2.40 µM respectively, suggesting a 6-fold and 24-fold selectivity, respectively, which was further confirmed by WB. Further, GT19077 and GT19506 were evaluated in panels of blood cancer and SCLC cell lines carrying Myc genetic alterations, as measured by. GT19077 was shown to selectively inhibit the proliferation of B-cell malignant cells with IGH/Myc genomic translocations in a panel of 14 blood cancer cell lines tested by CellTiter-Glo® Luminescent Cell Viability Assays (CTG). Consistently, GT19506 also selectively inhibited the proliferation of B-cell malignant cells in the panel of 28 blood cancer cell lines tested. Both compounds also inhibited the proliferation of SCLC cells in a panel of 18 SCLC cell lines tested (66% and 22%, respectively). Moreover, GT19077 inhibited c-Myc-driven tumor biology, including G2/M arrest and CD-47 and PD-L1 down-regulation. Finally, GT19077 and GT19506 demonstrated PK dependent c-Myc target engagement in HL-60 and Ramos xenograft tumor models. In conclusion, GT19077 and GT19506 are currently in Lead Optimization Phase, which demonstrate the desired selectivity of targeting Myc dependent B-cell malignancies and Myc-amplified SCLC, but sparing c-Myc in growth factor/cytokine regulated blood progenitor cells. The defined selectivity has enabled the ongoing in vivo efficacy studies, currently GT19077 and GT19506 have being evaluated in in vivo efficacy studies. Citation Format: Liandong Ma, Youzhi Tong, Qianxiang Zhou, Zhaohui Yang, Honghau Yan, Ye Chen, Ruo Xu, Jie Pan, Xiaoqi Gou, Weidong Qian, Jie Chen, Qi Sun, Luhua Lai. Discovery of GT19077, a c-Myc/Max protein-protein Interaction (PPI) small molecule inhibitor, and GT19506 a c-Myc PROTAC molecule, for targeting c-Myc-driven blood cancers and small cell lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1265.

Generation of two heterozygous GATA2 CRISPR/Cas9-edited iPSC lines, R398W and R396Q, for modeling GATA2 deficiency

Germline heterozygous GATA2 mutations underlie a complex disorder characterized by bone marrow failure, immunodeficiency and high risk to develop myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our understanding about GATA2 deficiency is limited due to the lack of relevant disease models. Here we generated high quality human induced pluripotent stem cell (iPSC) lines carrying two of the most recurrent germline GATA2 mutations (R389W and R396Q) associated with MDS, using CRISPR/Cas9. These hiPSCs represent an in vitro model to study the molecular and cellular mechanisms underlying GATA2 deficiency, when differentiated into blood progenitors.

Chromatin Regulator Mutations Are Drivers of Clonal Evolution in the Early Multipotent HSC Compartment of Primary Myelofibrosis

The definition of initiating genetic events remains up to date the main research quest in MPN disease biology. Identification of characteristic mutational clusters has shed more light on the complexity of various MPN genotypes, even though no specific genetic lesion or mutational order can be exclusively linked to the different MPN subtypes. Our aim is to determine the sequence of mutational clusters that orchestrate the expansion of aberrant clones in MPN with respect to the prime hematopoietic stem cell compartment.Genotyping for 54 MPN related gene mutations (100 MPN Patient cohort with myelofibrosis) indicated high incidence of mutations in chromatin regulator genes ASXL1 (40%) and EZH2 (14%). In 11% of the patients both ASXL1 and EZH2 gene mutations were detected, which correlated with significantly lower hemoglobin levels, higher leukocyte counts, higher numbers of additional mutations and higher risk score.In our former work we reported the identification of an expanded Lin-CD133+CD34+ neoplastic HSC population in patient peripheral blood that reproduces PMF parameters and fibrosis in vivo. Analysis of this aberrant HSC in the single cell level revealed the heterogeneity of the malignant clones and the mutational hierarchies that propagate myeloproliferation. From this analysis we report that ASXL1 and/or EZH2 mutations precede JAK2 signalling related genetic lesions (JAK2, CALR, MPL) or other epigenetic modifier mutational groups (DMT3A, TET2). Neoplastic stem cells bearing both ASXL1/EZH2 mutations exhibit higher levels of engraftment in vivo and limited erythroid colony output in vitro .In order to determine the exact contribution of chromatin regulator mutations with regard to clonal hematopoiesis determined by changes in prime HSC dynamics we established a sorting strategy for the isolation of early LT-HSC circulating in MF patient peripheral blood. By usage of 27 lineage specific and 6 additional HSC specific markers we sorted HSC on the apex of the hierarchy with the phenotype; 27xLin-CD34+CD38-CD90+CD93+CD49f+CD133+ and all classes of early and more committed progenitors from 11 patients with ASXL1 and/or EZH2 mutations. Our data indicate that both mutations emerge in the LT-HSC fraction (10/11 patients) dominate aberrant myelopoiesis and can be also detected in mature T cells (3/11 Patients). JAK2-V617F mutation was not detected in the early HSC fraction.The incidence of EZH2 mutations with or without the synergistic effect of ASXL1 mutations, is correlated with the expansion of morphologically aberrant erythroid progenitors in patient peripheral blood, which are found to be arrested in G0 phase of the cell cycle.Our results suggest that ASXL1 and EZH2 mutations are the drivers of clonal hematopoiesis in MPN and occur early at the multipotent HSC compartment. EZH2 mutations in PMF represent the first genetic cluster that correlates with aberrant erythropoiesis in these patients. DisclosuresNo relevant conflicts of interest to declare.

Aberrant Hedgehog Signaling in Myelodysplastic Syndrome Accelerates Leukemic Transformation Via Smoothened-Dependent and Smoothened-Independent Gli1 Activation

Introduction: Myelodysplastic syndromes (MDS) are malignant hematopoietic stem cell disorders with increased risk of transformation to secondary acute myeloid leukemia (sAML) that carries uniformly dismal prognosis. The exact mechanisms driving this process remain obscure. Aberrant Hedgehog (Hh) signaling has been demonstrated in a number of solid tumors and hematologic malignancies. An emerging body of literature suggests the importance of Hh signaling in leukemic transformation of MDS. Moreover, a recent Phase II clinical trial using Smoothened (SMO) inhibitor with low-dose chemotherapy resulted in significant prolongation of overall survival (Lancet et al. Blood 2016). Thus, we sought to examine the clinical impact of aberrant Hh activation in MDS and further explore the mechanism of Hh-driven leukemic transformation.Methods/Results:To determine the pathway activity, we quantified the level of GLI1 in CD34+ cells from patients with MDS and healthy controls in published databases (N=17 and N=159, respectively; GSE58831) and while absent in controls, GLI1 was significantly upregulated in MDS patients (p<0.05). Analysis of serial samples revealed Hh activation at disease progression in 67% of patients (4/6). In addition, GLI1 levels were significantly higher in poor-risk AML with complex cytogenetics comparing to favorable risk disease (p<0.001, TCGA).To further evaluate the impact of of Hh activation in MDS progression, we crossed Nup98-HoxD13 (NHD13) mice with mice conditionally expressing the constitutively active mutant of Smoothened (SmoM2). Double transgenic NHD13/SmoM2 mice succumbed to aggressive myeloid leukemia (median survival of NHD13/SmoM2 and NHD13 controls, 3 months vs. 13 months, respectively, p<0.0001) characterized by leukocytosis (p<0.05), splenomegaly (p<0.0001) and significant expansion of immature myeloid progenitors in peripheral blood (p<0.0001) and bone marrow (p<0.0001). Analysis of hematopoietic stem/progenitor (HSPC) compartment revealed expansion of phenotypic granulocyte macrophage progenitors (GMP) (6244 vs. 131487 mean GMPs/femur , in NHD13 v. NHD13/SmoM2 respectively, p<0.05). To determine whether Hh activation resulted in aberrant self-renewal in GMP compartment, we carried out an in vivo repopulation assay. While NHD13 GMPs showed only transient engraftment, characteristic of non-self-renewing progenitors, NHD13/SmoM2 GMPs robustly engrafted and significantly expanded over time, and all recipient animals developed aggressive leukemia within 2-3 months. The engraftment was sustained through secondary transplantation. Our results suggest that Hh activation resulted in abnormal self-renewal in normally non-self-renewing progenitors.To delineate the mechanism leading to aberrant activation of self-renewal we compared the transcriptome of GMPs isolated from NHD13/SmoM2 and NHD13 controls. In addition to expected Hh activation, Gene Set Enrichment Analysis (GSEA) showed enrichment in gene signatures associated with other developmental pathways such as Wnt or TGF-β signaling as well as EWS/FLI1-protooncogen signature. Interestingly, these pathways have been found to act via direct activation of GLI1 .Using human AML (TF-1) and MDS (MDS-L) cell lines we confirmed that GLI1 may be activated in the absence of SMO. GLI1 knock-down and/or inhibition with GANT61 resulted in growth arrest (p<0.05, both) and significantly lower clonogenic potential in vitro (p<0.05, both) more so than upstream SMO inhibition.Conclusions: Our data suggest that aberrant Hh/GLI1 signaling is frequent in MDS and marks disease progression and leukemic transformation. In some cases, GLI1 can be activated in a SMO-independent manner and therapeutic strategies focused on targeting downstream Gli transcription factors may be more effective than currently used Smo inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Targeting Mitochondrial Translation Overcomes Venetoclax Resistance in Acute Myeloid Leukemia (AML) through Activation of the Integrated Stress Response

Venetoclax is a highly specific BCL-2 inhibitor with promising activity against AML including leukemic stem cells (LSCs). However, venetoclax monotherapy in AML patients had limited clinical efficacy due to intrinsic or acquired drug resistance which has been attributed to the overexpression of other anti-apoptotic proteins including MCL-1 and BCL-xL. These findings highlight the importance of combination therapy to overcome resistance but the optimal strategy to achieving this goal is unclear.To address this issue, we generated a panel of highly resistant clones derived from the intrinsically sensitive MOLM-13 and MV-4-11 AML cell lines. Surprisingly, treatment with MCL-1 or BCL-xL inhibitors failed to overcome resistance despite higher expression of the anti-apoptotic proteins in resistant cells. To identify novel targets, we performed a genome-wide CRISPR knockout screen to find genes that upon inactivation, restore sensitivity to venetoclax. We transduced one of the resistant clones engineered to stably express Cas9 with a pooled lentiviral single-guide RNA (sgRNA) library that targets 17,661 protein-coding genes with 91,320 unique sgRNA sequences. Transduced cells were divided into 2 populations with one treated with venetoclax and the other untreated as control. The transduced cells were cultured for 29 days to negatively select sgRNAs that re-sensitized resistant cells to venetoclax. Cell samples were collected at regular intervals and subjected to next-generation sequencing of the sgRNA target region to quantify the abundance of each construct.We identified genes that were negatively selected in the presence of venetoclax using the MAGeCK algorithm. The top-ranked genes were highly enriched for gene ontology terms related to mitochondrial translation. We confirmed that RNAi-mediated knockdown of the top-ranked ribosomal subunit gene, DAP3, overcame venetoclax resistance. Pharmacologic inhibition of mitochondrial translation with a panel of protein synthesis inhibitor antibiotics similarly restored sensitivity in AML cell lines with intrinsic or acquired resistance. Tedizolid, a FDA-approved second generation oxazolidinone antibiotic, was the most effective in mediating this effect at clinically relevant concentrations. Furthermore, the combination of tedizolid and venetoclax targeted the LSC-enriched CD34+CD38- population in ex vivo -cultured primary AML samples. Importantly, normal cord blood hematopoietic stem and progenitor cells were more resistant to this combination suggestive of a wide therapeutic index .To decipher the mechanism by which tedizolid overcomes venetoclax resistance, we first profiled the expression of BCL-2 family members in total cellular and purified mitochondrial extracts. Antibiotic treatment did not affect total BCL-2, MCL-1, or BCL-xL protein levels but caused a substantial increase in BCL-2 levels in the mitochondrial fraction indicative of BCL-2 translocation to the organelle. Next, we confirmed that tedizolid treatment selectively reduced the expression of mitochondrial over nuclear-encoded proteins consistent with its inhibitory effect on mitochondrial translation. This mitonuclear protein imbalance has been shown to activate the integrated stress response (ISR). Tedizolid treatment activated this response as evidenced by an increase in eIF2-α phosphorylation and expression of ATF4, a master transcription factor regulator. To determine whether ISR activation is sufficient to overcome venetoclax resistance, we treated resistant cells with a small-molecule activator of PERK (CCT020312), which activates the ISR through direct eIF2-α phosphorylation, and observed re-sensitization to venetoclax.In summary, our results demonstrate that inhibition of mitochondrial translation, which activates ISR and triggers BCL-2 translocation to the mitochondria, is a novel approach to overcoming venetoclax resistance in AML cells. Our findings provide the rationale for exploring the use protein synthesis inhibitor antibiotics or direct ISR activators in combination with venetoclax for the treatment of AML or other malignancies. DisclosuresNo relevant conflicts of interest to declare.