Myeloid Maturation Research Articles

Complementary and countervailing actions of Jak2 and Ikk2 in hematopoiesis in mice

Hyperactivation of JAK2 kinase is a unifying feature of human Ph- myeloproliferative neoplasms (MPNs), most commonly due to the JAK2 V617F mutation. Mice harboring a homologous mutation in the Jak2 locus exhibit a phenotype resembling polycythemia vera. NFκB pathway hyperactivation is present in myeloid neoplasms, including MPNs, despite scarcity of mutations in NFκB pathway genes. To determine the impact of NFκB pathway hyperactivation in conjunction with Jak2 V617F, we utilized Ikk2 (Ikk2-CA) mice. Pan-hematopoietic Ikk2-CA alone produced depletion of hematopoietic stem cells and B cells. When combined with the Jak2 V617F mutation, Ikk2-CA rescued the polycythemia vera phenotype of Jak2 V617F. Likewise, Jak2 V617F ameliorated defects in hematopoiesis produced by Ikk2-CA. Single-cell RNA sequencing of hematopoietic stem and progenitor cells revealed multiple genes antagonistically regulated by Jak2 and Ikk2, including subsets whose expression was altered by Jak2 V617F and/or Ikk2-CA but partly or fully rectified in the double mutant. We hypothesize that Jak2 promotes hematopoietic stem cell population self-renewal, whereas Ikk2 promotes myeloid lineage differentiation, and biases cell fates at several branch points in hematopoiesis. Jak2 and Ikk2 both regulate multiple genes affecting myeloid maturation and cell death. Therefore, the presence of dual Jak2 and NFκB hyperactivation may present neomorphic therapeutic vulnerabilities in myeloid neoplasms.

Systemic administration of STING agonist promotes myeloid cells maturation and antitumor immunity through regulating hematopoietic stem and progenitor cell fate.

STING is a pivotal mediator of effective innate and adaptive anti-tumor immunity; however, intratumoral administration of STING agonists have shown limited therapeutic benefit in clinical trials. The systemic effect of the intravenous delivery of STING agonists in cancer is not well-defined. Here, we demonstrated that systemic administration of STING agonist inhibited melanoma growth, improved inflammatory effector cell infiltration, and induced bone marrow mobilization and extramedullary hematopoiesis, causing widespread changes in immune components in the peripheral blood. The systemically administered STING agonist promoted HSC expansion and influenced lineage fate commitment, which was manifested as the differentiation of HSPCs was skewed toward myeloid cells at the expense of B-cell lymphopoiesis and erythropoiesis. Transcriptome analysis revealed upregulation of myeloid lineage differentiation-related and type I interferon-related genes. This myeloid-biased differentiation promoted the production and maturation of myeloid cells toward an activated phenotype. Furthermore, depletion of Gr-1+ myeloid cells attenuated the anti-tumor immunity of STING agonist. Our findings reveal the anti-tumor mechanism of systemic administration of STING agonist that involves modulating HSPC differentiation and promoting myeloid cells maturation. Our study may help explain the limited clinical activity of STING agonists administered intratumorally.

Targeting miR-223 enhances myeloid-derived suppressor cell suppressive activities in multiple sclerosis patients.

Multiple sclerosis (MS) is an incurable autoimmune inflammatory demyelinating disease of the central nervous system. Several MS medications can modify disease course through effects on adaptive immune cells, while drugs targeting innate immunity are under investigation. Myeloid-derived suppressor cells (MDSCs) which arise during chronic inflammation, are defined by their T-cell immunosuppressive functions. MiR-223 modulates myeloid cell maturation and expansion, including MDSCs. MDSCs isolated from healthy controls (HC) and people with MS (pwMS) were co-cultured with CD4+ T-cells to study their immunosuppressive activities in vitro. Cytokines and chemokines concentration were evaluated by Luminex assay in the serum of HC, pwMS, and other neuroinflammatory diseases and correlated with MDSC activities. MDSC suppressive functions are dysregulated in pwMS compared to HC, which was reversed by glucocorticoids (GC). GC specifically downregulated miR-223 levels in MDSCs and increased the expression of STAT3. In vitro assay showed that miR-223 inhibition enhanced MDSC suppressive activity, STAT3 dependently. By multiple linear regression analysis, we demonstrated that MDSC phosphorylated STAT3 was correlated with serum GM-CSF in HC and pwMS. These results suggest that miR-223 could be a therapeutic target for enhancing MDSC's suppressive activities as an alternative to GC.

ThPOK is a critical multifaceted regulator of myeloid lineage development.

The transcription factor ThPOK (encoded by Zbtb7b) is well known for its role as a master regulator of CD4 lineage commitment in the thymus. Here, we report an unexpected and critical role of ThPOK as a multifaceted regulator of myeloid lineage commitment, differentiation and maturation. Using reporter and knockout mouse models combined with single-cell RNA-sequencing, progenitor transfer and colony assays, we show that ThPOK controls monocyte-dendritic cell versus granulocyte lineage production during homeostatic differentiation, and serves as a brake for neutrophil maturation in granulocyte lineage-specified cells through transcriptional regulation of lineage-specific transcription factors and RNA via altered messenger RNA splicing to reprogram intron retention.

Secondary-Type Mutations in Acute Myeloid Leukemia: Updates from ELN 2022.

The characterization of the molecular landscape and the advent of targeted therapies have defined a new era in the prognostication and treatment of acute myeloid leukemia. Recent revisions in the European LeukemiaNet 2022 guidelines have refined the molecular, cytogenetic, and treatment-related boundaries between myelodysplastic neoplasms (MDS) and AML. This review details the molecular mechanisms and cellular pathways of myeloid maturation aberrancies contributing to dysplasia and leukemogenesis, focusing on recent molecular categories introduced in ELN 2022. We provide insights into novel and rational therapeutic combination strategies that exploit mechanisms of leukemogenesis, highlighting the underpinnings of splicing factors, the cohesin complex, and chromatin remodeling. Areas of interest for future research are summarized, and we emphasize approaches designed to advance existing treatment strategies.

Contrariety of Human Bone Marrow Mesenchymal Stromal Cell Functionality in Modulating Circulatory Myeloid and Plasmacytoid Dendritic Cell Subsets.

Mesenchymal Stromal Cells (MSCs) derived from bone marrow are widely tested in clinical trials as a cellular therapy for potential inflammatory disorders. The mechanism of action of MSCs in mediating immune modulation is of wide interest. In the present study, we investigated the effect of human bone-marrow-derived MSCs in modulating the circulating peripheral blood dendritic cell responses through flow cytometry and multiplex secretome technology upon their coculture ex vivo. Our results demonstrated that MSCs do not significantly modulate the responses of plasmacytoid dendritic cells. However, MSCs dose-dependently promote the maturation of myeloid dendritic cells. Mechanistic analysis showed that dendritic cell licensing cues (Lipopolysaccharide and Interferon-gamma) stimulate MSCs to secret an array of dendritic cell maturation-associated secretory factors. We also identified that MSC-mediated upregulation of myeloid dendritic cell maturation is associated with the unique predictive secretome signature. Overall, the present study demonstrated the dichotomy of MSC functionality in modulating myeloid and plasmacytoid dendritic cells. This study provides clues that clinical trials need to investigate if circulating dendritic cell subsets in MSC therapy can serve as potency biomarkers.

Abstract A19: Targeting epigenetic regulators to override cellular identity programs and induce therapeutic differentiation in MLL-rearranged acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is a poor-prognosis disease that is universally characterized by a prominent differentiation block. Chemotherapeutic regimens and disease outcomes have not changed in decades, and there is great need for novel therapeutic treatments and approaches. Differentiation therapy is a fundamentally different therapy that aims to reactivate latent maturation programs to induce proliferation arrest and apoptosis. This approach is curative in the promyelocytic AML subtype but not others. It is therefore critical to identify the enforcers of differentiation arrest in non-APL AML. Here, we performed a cell-fate selection CRISPR screen for chromatin factors whose inhibition promotes AML cell differentiation. We found that both genetic and chemical inhibition of the histone acetyltransferase KAT6A induces myeloid maturation, most prominently in MLL-rearranged AML. KAT6A loss markedly reduces self-renewal and extinguishes the proliferative capacity of AML cells. Through ChIP-seq, RNA-seq, and clinical dataset analyses, we found that KAT6A is the likely initiator of a newly-described "writer-reader" module that drives oncogene expression in AML. KAT6A catalyzes promoter H3K9 acetylation, which is bound by the H3K9ac reader ENL, leading to Super-elongation complex recruitment and release of paused RNA PolII at oncogenic loci such as MYC. KAT6A is elevated in human AMLs compared to matched normal tissue, and its downregulation correlates with monocytic differentiation in clinical AML datasets. These findings suggest the potential of targeting KAT6A and/or ENL to disrupt this writer-reader module and ablate downstream MYC transcriptional programs in MLL-rearranged AML. This strategy may be viable in the near future, as a recently developed KAT6A/B inhibitor has already entered clinical trials for breast cancer. In follow-up work, we performed differentiation-specific "synergy screens," in which we delivered sgRNA libraries to U937 cells and ER-Hoxb8 cells treated with or without an LSD1 inhibitor (LSD1i). We identified multiple genes whose knockout synergized with LSD1i treatment to induce complete, terminal differentiation with low toxicity. Genetic results were confirmed with small molecule inhibitors of selected hits, and the most synergistic drug combination was pursued further. We found that AML cells dependent on hyperactivity of both Hoxa9 and Meis1 are most sensitive to this drug combination. Preliminary data suggest that LSD1i treatment alone fails to downregulate Meis1 expression, and that the ability of the second drug to fully ablate Meis1 is the likely mechanism underlying the observed synergy. Importantly, the second drug is also in clinical trials and will be discussed in the abstract presentation. Altogether, this work advances our understanding of the AML differentiation block and nominates a specific drug combination for AML differentiation therapy. Citation Format: Andres Blanco. Targeting epigenetic regulators to override cellular identity programs and induce therapeutic differentiation in MLL-rearranged acute myeloid leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A19.

The function and regulation of CCAAT/enhancer binding protein ε

In recent years, studies on the structure, function, and regulation of the C/EBPε gene have become an essential topic in the field of many diseases. CCAAT/enhancer-binding protein ε (C/EBPε) is the fifth member of the transcription factor CCAAT/C/EBP family of transcription factors. It plays crucial roles in cell proliferation, differentiation, immunity, energy metabolism, and hematopoiesis. C/EBPε plays essential roles in regulating the hematopoietic system, including myeloid cell development and maturation, participation in the body’s immune responses, and prevention of infections. C/EBPε function is regulated by phosphorylation, acetylation, methylation, and other types of genes. This review related to C/EBPε structure, function and regulation provides a theoretical basis for subsequent research in this area. C/EBPε is an emerging therapeutic target and thus provides new strategies for disease prevention and control.

Asxl1 deletion disrupts MYC and RNA polymerase II function in granulocyte progenitors.

Mutations in the gene Additional Sex-Combs Like 1 (ASXL1) are recurrent in myeloid malignancies as well as the pre-malignant condition clonal hematopoiesis, where they are universally associated with poor prognosis. However, the role of ASXL1 in myeloid lineage maturation is incompletely described. To define the role of ASXL1 in myelopoiesis, we employed single cell RNA sequencing and a murine model of hematopoietic-specific Asxl1 deletion. In granulocyte progenitors, Asxl1 deletion leads to hyperactivation of MYC and a quantitative decrease in neutrophil production. This loss of granulocyte production was not accompanied by significant changes in the landscape of covalent histone modifications. However, Asxl1 deletion results in a decrease in RNAPII promoter-proximal pausing in granulocyte progenitors, indicative of a global increase in productive transcription. These results suggest that ASXL1 inhibits productive transcription in granulocyte progenitors, identifying a new role for this epigenetic regulator in myeloid development.

NK cells reduce anergic T cell development in early-stage tumors by promoting myeloid cell maturation.

Studies of NK cells in tumors have primarily focused on their direct actions towards tumor cells. We evaluated the impact of NK cells on expression of homing receptor ligands on tumor vasculature, intratumoral T cell number and function, and T cell activation in tumor draining lymph node. Using an implantable mouse model of melanoma, T cell responses and homing receptor ligand expression on the vasculature were evaluated with and without NK cells present during the early stages of the tumor response by flow cytometry. NK cells in early-stage tumors are one source of IFNγ that augments homing receptor ligand expression. More significantly, NK cell depletion resulted in increased numbers of intratumoral T cells with an anergic phenotype. Anergic T cell development in tumor draining lymph node was associated with increased T-cell receptor signaling but decreased proliferation and effector cell activity, and an incomplete maturation phenotype of antigen presenting cells. These effects of NK depletion were similar to those of blocking CD40L stimulation. We conclude that an important function of NK cells is to drive proper APC maturation via CD40L during responses to early-stage tumors, reducing development of anergic T cells. The reduced development of anergic T cells resulting in improved tumor control and T cell responses when NK cells were present.

Inhibiting the biogenesis of myeloid-derived suppressor cells enhances immunotherapy efficacy against mammary tumor progression.

While immune checkpoint inhibitors (ICIs) have transformed the therapeutic landscape in oncology, they are effective in select subsets of patients. Efficacy may be limited by tumor-driven immune suppression, of which 1 key mechanism is the development of myeloid-derived suppressor cells (MDSCs). A fundamental gap in MDSC therapeutics is the lack of approaches that target MDSC biogenesis. We hypothesized that targeting MDSC biogenesis would mitigate MDSC burden and bolster tumor responses to ICIs. We tested a class of agents, dihydroorotate dehydrogenase (DHODH) inhibitors, that have been previously shown to restore the terminal differentiation of leukemic myeloid progenitors. DHODH inhibitors have demonstrated preclinical safety and are under clinical study for hematologic malignancies. Using mouse models of mammary cancer that elicit robust MDSC responses, we demonstrated that the DHODH inhibitor brequinar (a) suppressed MDSC production from early-stage myeloid progenitors, which was accompanied by enhanced myeloid maturation; (b) augmented the antitumor and antimetastatic activities of programmed cell death 1-based (PD-1-based) ICI therapy in ICI-resistant mammary cancer models; and (c) acted in concert with PD-1 blockade through modulation of MDSC and CD8+ T cell responses. Moreover, brequinar facilitated myeloid maturation and inhibited immune-suppressive features in human bone marrow culture systems. These findings advance the concept of MDSC differentiation therapy in immuno-oncology.

Fibroblast inflammatory priming determines regenerative versus fibrotic skin repair in reindeer

Adult mammalian skin wounds heal by forming fibrotic scars. We report that full-thickness injuries of reindeer antler skin (velvet) regenerate, whereas back skin forms fibrotic scar. Single-cell multi-omics reveal that uninjured velvet fibroblasts resemble human fetal fibroblasts, whereas back skin fibroblasts express inflammatory mediators mimicking pro-fibrotic adult human and rodent fibroblasts. Consequently, injury elicits site-specific immune responses: back skin fibroblasts amplify myeloid infiltration and maturation during repair, whereas velvet fibroblasts adopt an immunosuppressive phenotype that restricts leukocyte recruitment and hastens immune resolution. Ectopic transplantation of velvet to scar-forming back skin is initially regenerative, but progressively transitions to a fibrotic phenotype akin to the scarless fetal-to-scar-forming transition reported in humans. Skin regeneration is diminished by intensifying, or enhanced by neutralizing, these pathologic fibroblast-immune interactions. Reindeer represent a powerful comparative model for interrogating divergent wound healing outcomes, and our results nominate decoupling of fibroblast-immune interactions as a promising approach to mitigate scar.

Abstract B027: The transcription factor HIF2alpha partakes in the differentiation block of acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is characterized by epigenetic silencing of differentiation genes and accumulation of aberrant myeloid cells arrested at different stages of myeloid development. The combination of epigenetic and differentiation therapies represents an attractive opportunity for AML patients by promoting chromatin remodeling at differentiation genes and induction of terminal maturation and leukemia debulking. The aim of our work is to define the function of the hypoxia inducible transcription factor HIF2alpha in the pathogenesis of AML. By its genetic inactivation and pharmacological inhibition in AML cell lines and patient-derived xenograft models, we found that HIF2alpha acts as a novel regulator of the AML differentiation block and promotes leukemia progression. Gene expression profiling of HIF2alpha-dependent transcriptome revealed that HIF2alpha mainly promotes the expression of genes involved in transcriptional modulation and epigenetic modifications, whilst inhibiting genes of myeloid maturation and activation. Mechanistically, inhibition of HIF2alpha results in decreased global levels of the heterochromatin marker H3K27me3, which is specifically deposited by the catalytic subunit EZH2 of the Polycomb repressive complex 2. Intriguingly, we found that HIF2alpha and EZH2 cooperate at favoring EZH2-mediated deposition of H3K27me3 at reverse hypoxia responsive elements in the regulatory regions of myeloid maturation genes. Additionally, we demonstrate that HIF2alpha is positively regulated by the pro-differentiation agent all-trans retinoic acid (ATRA), and its inhibition cooperates with ATRA in triggering AML cell differentiation. In conclusion, we provide new mechanistic insights into the role of HIF2alpha in the pathogenesis of AML, by promoting an undifferentiated state via EZH2-mediated epigenetic silencing of myeloid differentiation genes. Importantly, small molecule inhibitors of HIF2alpha have been recently generated and are tested for solid cancers. Therefore, HIF2alpha inhibition may open new therapeutic avenues for AML patients and add therapeutic value to ATRA-based therapies by interrupting HIF2alpha upregulation and promoting an epigenetic state permissive to maturation of leukemic blasts and leukemia exhaustion. Citation Format: Daniela Magliulo, Carolina Caserta, Serena Belluschi, Cristina Fracassi, Kety Giannetti, Raffaella Pini, Ettore Zapparoli, Stefano Beretta, Eleonora Draghi, Marco J. Morelli, Raffaella Di Micco, Bernhard Gentner, Luca Vago, Rosa Bernardi. The transcription factor HIF2alpha partakes in the differentiation block of acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B027.

CDDO Induces Differentiation through PTEN Expression in HL-60 Cells

Acute promyelocytic leukemia (APL) represents a group of haematological malignancies characterized by blocked differentiation. Cell differentiation has been as a strong target of treatment of APL. Peroxisome proliferator-activated receptors gamma (PPAR-γ) play an important role in the differentiation of APL cell lines. Previous study has shown that PPAR-γ ligands induce neutrophil/ monocytic differentiation in human acute promyelocytic leukemia HL-60 cells. It has recently been shown that the novel synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) induces differentiation in leukemia HL-60 cells. In this study we established a CDDO-induced differentiation model for the treatment of APL. We evaluated for the surface antigen expression associated with myeloid maturation, CD11b and CD14 by flow cytometric analysis and RT-PCR. We demonstrated that CD11b and CD14 increased as CDDO -induced differentiation. In addition, CDDO induced a HL-60 differentiation through the regulation of PTEN expression. PTEN also has various cellular function such as cell differentiation. PPAR-γ could synergistically up-regulate PTEN in human leukemia cells and consequently stimulate the differentiation of these cells We confirmed that suppression of PTEN with the inhibitor (SF1760) caused reduction of differentiated HL-60 cells. Our results suggest PPAR-γ an important role in controlling the differentiation of leukemia cells via the regulation of PTEN. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

SYK Inhibitors, Entospletinib and Lanraplenib, Show Potent Anti-Leukemic Activity in Combination with Targeted Agents

Introduction: Spleen tyrosine kinase (SYK) acts as a key integrator of cellular signaling from surface contact and receptor tyrosine kinase receptors containing an immunoreceptor tyrosine-based activation motif (ITAM). In acute myeloid leukemia (AML), SYK serves as a relay to an oncogenic transcriptional regulatory network (TRN) linked to NPM1, HOXA9, and MEIS1 helping to suppress myeloid progenitor maturation and promote proliferation. The selective, oral SYK inhibitor, entospletinib (ENTO), has demonstrated clinical activity in HOXA9/MEIS1-driven AML with acceptable tolerability when combined with intensive induction chemotherapy. A global phase 3 trial (AGILITY, NCT05020665) is currently underway, evaluating the combination of ENTO with intensive induction/consolidation chemotherapy in patients with treatment-naive NPM1-mutated (NPM1m) AML with measurable residual disease as the primary endpoint. Lanraplenib (LANRA) is a next-generation SYK inhibitor with similar potency, enhanced selectivity, and more favorable pharmacologic properties that is currently being evaluated in combination with gilteritinib in patients with relapsed or refractory (R/R) FLT3-mutated AML (NCT05028751). Results: ENTO exhibits anti-proliferative activity in NPM1m AML with associated HOXA9/MEIS1 dysregulation in ex vivo drug sensitivity studies. We hypothesized that SYK inhibition could synergize with other targeted therapies to enhance anti-leukemic activity at different nodes in this pathway. To address this hypothesis, the FLT3 internal tandem duplication/MLL-rearranged cell lines, MOLM13 and MV411, were treated with increasing doses of either LANRA or ENTO in combination with a small molecule MLL-Menin inhibitor (SNDX5613). Synergistic anti-proliferative effects were observed across a broad range of concentrations. No combinatorial or single-agent SNDX5613 activity was seen in the KASUMI-1 AML cell line, which lacks an MLL rearrangement. Phenotypic studies using flow cytometry were performed to assess the mechanistic response for SYK inhibition with LANRA in combination with SNDX5613. LANRA was found to increase the percentage of CD11b-positive cells and mean fluorescence intensity, while SNDX5613 led to a marked increase in CD14. In contrast to either single agent, the combination triggered early apoptosis and cell death, suggesting a more complete blockade of the HOXA9/MEIS1 transcriptional program through synergistic inhibition by orthogonal mechanisms. Furthermore, treatment of leukemic cells with either ENTO or LANRA inhibited SYK auto-phosphorylation in a dose-dependent manner. We also explored the potential for synergistic activity with gilteritinib (FLT3 inhibitor) and venetoclax (BCL2 inhibitor) using patient-derived AML isolates ex vivo based on the potential for SYK signaling cross talk with FLT3 itself and survival pathways. We found strong synergistic anti-proliferative activity for the combination of LANRA with either gilteritinib or venetoclax. Patient-derived xenograft (PDX) studies also demonstrated deeper reductions in leukemic burden in the peripheral blood, liver, and bone marrow after 28 days of treatment. Follow-up PDX studies assessing overall survival and pharmacodynamic activity are ongoing using an optimized dosing regimen and schedule. Summary/Conclusion: SYK is a promising therapeutic target for genetically defined subsets of AML. Two highly selective SYK inhibitors are currently undergoing clinical testing in different but complementary clinical settings. Given its central role as a modulator of leukemogenic signaling, SYK inhibition has the potential to synergize with other targeted therapies in AML. In preclinical studies, LANRA-mediated SYK inhibition shows compelling anti-proliferative and pro-apoptotic activity in combination with gilteritinib, venetoclax, and SNDX5613 in genetically defined subsets of AML.

MiR-196b-TLR7/8 Signaling Axis Regulates Innate Immune Signaling and Myeloid Maturation in DNMT3A-Mutant AML.

DNMT3A mutations confer a poor prognosis in acute myeloid leukemia (AML), but the molecular mechanisms downstream of DNMT3A mutations in disease pathogenesis are not completely understood, limiting targeted therapeutic options. The role of miRNA in DNMT3A-mutant AML pathogenesis is understudied. DNA methylation and miRNA expression was evaluated in human AML patient samples and in Dnmt3a/Flt3-mutant AML mice. The treatment efficacy and molecular mechanisms of TLR7/8-directed therapies on DNMT3A-mutant AML were evaluated in vitro on human AML patient samples and in Dnmt3a/Flt3-mutant AML mice. miR-196b is hypomethylated and overexpressed in DNMT3A-mutant AML and is associated with poor patient outcome. miR-196b overexpression in DNMT3A-mutant AML is important to maintain an immature state and leukemic cell survival through repression of TLR signaling. The TLR7/8 agonist resiquimod induces dendritic cell-like differentiation with costimulatory molecule expression in DNMT3A-mutant AML cells and provides a survival benefit to Dnmt3a/Flt3-mutant AML mice. The small molecule bryostatin-1 augments resiquimod-mediated AML growth inhibition and differentiation. DNMT3A loss-of-function mutations cause miRNA locus-specific hypomethylation and overexpression important for mutant DNMT3A-mediated pathogenesis and clinical outcomes. Specifically, the overexpression of miR-196b in DNMT3A-mutant AML creates a novel therapeutic vulnerability by controlling sensitivity to TLR7/8-directed therapies.

034 Tissue specificity of dendritic cells supersedes subset identity

To prevent acute life-threatening auto-inflammation/autoimmune of the tissues, our immune system must mount protective immunity, and rapidly resolve inflammation. Dendritic cells (DCs) are specialized immune sentinels that maintain immune homeostasis by efficiently regulating the balance between protective immunity and tolerance to self-antigens. It is critical to understand how tissue DCs subsets and their tissue specificity are governed to mediate protective or tolerant immunity. By assaying chromatin accessibility of LN resident (cDC1 vs cDC2) and migratory DCs (migDC1 vs migDC2) genome-wide through ATACseq analysis, together with our prior lab work using RNAseq analysis, we found that tissue myeloid maturation and migration programing supersedes subset diversification, and is enforced at the level of the chromatin.

Continuous treatment with abemaciclib leads to sustained and efficient inhibition of breast cancer cell proliferation.

Abemaciclib is an oral, selective cyclin-dependent kinase 4 & 6 inhibitor (CDK4 & 6i), approved for hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2–) advanced breast cancer (ABC) as monotherapy for endocrine refractory disease, and with endocrine therapy (ET) for initial treatment and after progression on ET. Abemaciclib has also shown clinical activity in combination with ET in patients with high risk early BC (EBC). Here, we examined the preclinical attributes of abemaciclib and other CDK4 & 6i using biochemical and cell-based assays. In vitro, abemaciclib preferentially inhibited CDK4 kinase activity versus CDK6, resulting in inhibition of cell proliferation in a panel of BC cell lines with higher average potency than palbociclib or ribociclib. Abemaciclib showed activity regardless of HER2 amplification and phosphatidylinositol 3-kinase (PI3KCA) gene mutation status. In human bone marrow progenitor cells, abemaciclib showed lower impact on myeloid maturation than other CDK4 & 6i when tested at unbound concentrations similar to those observed in clinical trials. Continuous abemaciclib treatment provided profound inhibition of cell proliferation, and triggered senescence and apoptosis. These preclinical results support the unique efficacy and safety profile of abemaciclib observed in clinical trials.

P748: SINGLE CELL GENOTYPING OF MATCHED BONE MARROW AND PERIPHERAL BLOOD CELLS IN TREATMENT NAIVE AND AZA-TREATED MDS AND CMML

Background: Somatic mutations in hematopoietic stem cells (HSCs) are a central pathogenic event in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) where they induce proliferative advantages and impaired differentiation with subsequent cytopenias in peripheral blood (PB). Patients with high-risk disease who are ineligible for allogenic stem cell transplantation are treated with hypomethylating agents, including 5-azacytidine (AZA). AZA treatment can improve PB counts and delay progression to Akute Myeloid Leukemia. However, AZA response does not require eradication of mutated HSCs and may be related to improved differentiation capacity of mutated hematopoietic stem and progenitor cells (HSPCs). However, the contribution of mutated HSPCs to steadystate hematopoiesis in MDS and CMML is unclear. Aims: In this project, we aimed to determine the distribution of individual subclones and even wild-type cells within the HSPC-compartment and the contribution of these subclones and wild-type cells to effective hematopoiesis in patients with MDS or CMML. Methods: We used a combination of index sorting and single cell genotyping to characterize the somatic mutations of individual stem- and progenitor cells and matched high-turnover circulating cells in three patients, one treatment naïve and two AZA-treated. Using this approach, myeloid driver mutations previously detected in a traditional approach were tracked in HSC/multipotent progenitors (MPP), MDS stem cells MDS-SC, progenitor cells (common myeloid progenitor (CMP), granulocyte monocyte progenitor (GMP), megakaryocyte erythroid progenitor (MEP)), as well as in monocytes, neutrophils and naïve B cells (nBC). Results: Patient H198302 and Patient H198303 both had CMML and had been treated with AZA for ̴10 years with complete response. Patient H198304 had MDS-EB1 and has never been treated with hypomethylating agents. For each patient, the figure shows the proportions of cells across the haematopoietic hierarchy carrying zero, one, two, three, or four mutations in the specified alleles. Only very small numbers of HSPCs carrying no mutation could be detected. In all three patients, most stem and progenitor cells carried a majority of tracked mutations, with no major differences between stem cells, progenitor cells and MDS-stem cells. In one patient, H198304, MDS-stem cells had a higher proportion of cells with 3 or 4 mutations detected. Overall, in all three patients, a similar frequency of mutations was observed in differentiated monocytes and neutrophils with a substantial proportion of the circulating cells derived from highly mutated progenitors. One notable exception were nBCs in Patient H198304, which were mostly wild-type, suggesting that the small wildtype HSC population is the predominant origin of nBCs in this individual. Analysis of additional lymphoid populations in PB from this patient revealed that naïve T, but not NK cells were also predominantly wild-type, suggesting specific impairment of B- and T-lineage maturation in the mutated cells. Image:Summary/Conclusion: In conclusion, attrition of highly mutated cells during myeloid maturation was not observed in any of the three patients, irrespective of HMA therapy. This suggests that in vivo, highly mutated stem and progenitor cells retain the capacity to differentiate to mature myeloid and in some patients mature lymphoid cells and contribute significantly to circulating blood cells in MDS and CMML, prior to and following AZA treatment.

P1051: A PHASE 2 STUDY OF IMG-7289 (BOMEDEMSTAT) IN PATIENTS WITH ADVANCED MYELOFIBROSIS

Background: Lysine-specific demethylase-1 (LSD1) is an activity critical for the self-renewal of malignant myeloid cells and maturation of megakaryocytes, cells central to the pathogenesis of MF. Bomedemstat is an orally active LSD1 inhibitor that in mouse models ameliorated the hallmarks of MPNs and improved survival (Kleppe et al. 2015; Jutzi et al. 2018). Aims: IMG-7289-CTP-102 is an ongoing, global, open-label Phase 2 study evaluating bomedemstat dosed QD in MF patients (NCT03136185). Key eligibility criteria include patients intolerant, refractory, resistant, or inadequately controlled by approved therapy, and platelet count ≥100 x 109/L. Key objectives are safety and reduction of spleen volume (SVR) by MRI/CT and total symptoms scores (TSS) using the MPN-SAF instrument. Methods: Serial bone marrow (BM) biopsies and imaging studies are read centrally. 261 genes are serially sequenced to quantify changes in the allelic frequencies of mutations (MAF) and identify new mutations. The starting dose is 0.6 mg/kg/d titrating, as needed, to a platelet count of 50-75x109/L. Results: At 89 patients, the study is now fully enrolled: 46% primary MF, 33% post-essential thrombocythaemia-MF, 21% post-polycythaemia vera-MF. Median age is 68 (35-88) with 52% males. Prior treatment with ruxolitinib was reported in 83% (74/89); 46% had also received at least 1 additional treatment. 30% of patients (27/89) had received ≥1 RBC transfusion prior to dosing. By IPSS, 53% were high-risk, 40% int.-2, and 7% int.-1. At screening (N=103), sequencing to a mean depth of >1000 bp, JAK2 was mutated in 69%, CALR in 21%, MPL in 6%; 59% had ≥2 mutations of which 71% were high-molecular risk mutations in ASXL1, IDH1/2, EZH2, U2AF1, TP53 and/or SRSF2. At data cutoff (3 Feb 2022), the median duration of treatment is 28 weeks (2-131). Of patients for whom TSS data is available at 24 weeks in those with baseline values ≥20, 72% (18/25) had a reduction in TSS; 24% (6/25) reported a ≥50% reduction. Of patients evaluable for SVR (N=50), 64% had a reduction in spleen volume from baseline; in 28%, SVR was ≥20%. Of evaluable patients (N=41), 90% had stable (∆ <±1.0 g/dL) or improved (≥1.0 g/dL) hemoglobin. Of patients with BM fibrosis scoring post-baseline (N=52), 31% improved by 1 grade and 50% were stable. CCL5 and S100A8/A9 cytokines were elevated at baseline in 50% (16/32) and 78%, respectively; at Day 84, 81% and 68%, respectively, showed reductions of at least 10% and of those, 100% (CCL5) and 47% (S100A8/A9) normalized. In follow-up sequencing at around Week 24, of 60 mutant alleles in 32 patients, the mean MAF fell by 39% in 48%. JAK2 MAFs fell by 31% [SD 5%] in 46% (N=24); ASXL1 MAFs fell 40% in 71%. Clones with JAK2 and/or ASXL1 mutations were most affected. No new mutations have been identified and no patient has transformed to AML. The most common non-hematologic AEs reported by patients was dysgeusia in 36% (32/90) and diarrhoea in 34% (31/90). All dysgeusia events were grade 1/2 and 1 led to treatment discontinuation. Of 14 related SAEs, 4 were Grade 2, 9 Grade 3 and 1 Grade 4 (thrombocytopenia). Twenty-nine patients remain on bomedemstat. Early terminations due to AEs occurred in 18 (20%) patients (9 related to bomedemstat), and 13 discontinued for other reasons. There have been no safety signals, DLTs, or deaths related to drug. Summary/Conclusion: In patients with advanced MF, bomedemstat alone had an acceptable tolerability profile, relieved symptoms, reduced spleen volume and mutation burden while improving fibrosis and anemia without safety signals.