Circular RNAs are a novel class of RNA transcripts, which regulate important cellular functions in health and disease. Herein, we report on the functional relevance of circPCMTD1 in BCR/ABL1-positive myeloid leukemias. In screening experiments, we found that circPCMTD1 depletion strongly inhibited the proliferative capacity of leukemic cells with BCR/ABL1 translocations. RNA sequencing and mass cytometry experiments identified aberrant activation of the DNA damage response (DDR) pathway as a downstream effect of circPCMTD1 depletion. DNA fiber assays, Comet assays and profiling of DDR markers (phospho-H2AX, phospho-CHK1, etc.) further underscored the pronounced effect of circPCMTD1 depletion in increasing genotoxic stress and inhibiting leukemic cell growth. circPCMTD1 targeting also led to aberrant DDR activation in leukemia patient blasts with BCR/ABL1 translocations. In in vivo experiments, circPCMTD1 knock-down prolonged the survival of mice engrafted with BCR/ABL1-positive leukemia cells. Mechanistically, we found that circPCMTD1 is enriched in the cytoplasm and associates with the ribosomes of leukemic blasts. We detected a cryptic open reading frame within the circPCMTD1 sequence and found that circPCMTD1 generates a 127 amino-acid peptide product (cPCMTD1-127aa). Using a custom-produced antibody, we found that the cPCMTD1-127aa interacts with the BCR/ABL1 oncoprotein, as well as with the BLM, TOP3A and RMI1 proteins, which form the BTR complex and regulate DNA repair and genome stability. cPCMTD1-127aa enhanced BTR complex formation, thereby increasing cellular tolerance to genotoxic stress. In summary, we identify and characterize circPCMTD1 as a molecular vulnerability and potential therapeutic target in BCR/ABL1-positive leukemias.

Obesity expands myeloid progenitors, myelopoiesis and increases the production of monocytes. While weight loss (WL) alleviates aspects of this inflammatory dysregulation, it is not known whether GLP-1 receptor agonists or other traditional modalities of WL differentially modify hematopoietic stem/progenitor cells (HSPCs), hematopoiesis, or inflammatory cell production. To test this, we compared the hematopoietic compartment in lean, obese and weight-reduced mice from tirzepatide treatment and caloric restriction (CR) implemented to match the body weight in both groups. At equal WL, we found CR induced multilineage cytopenias, whereas tirzepatide preserved blood lineages while specifically reducing classical Ly6Chi CCR2+ monocytes. To define the mechanisms underlying these changes we performed single-cell mRNA sequencing of bone marrow HSPCs and mature mononuclear blood cells. CR-HSPCs suppressed gene sets associated with nutrient sensing, proliferation and oxidative phosphorylation (OXPHOS) and exhibited lower inferred cell cycle activity, whereas tirzepatide-HSPCs attenuated these changes. Unlike CR, we found that across progressively differentiated cells from HSPCs to mature blood monocytes, tirzepatide increasingly suppressed OXPHOS and simultaneously shifted the maturation spectrum away from classical monocytes. Following six weeks of tirzepatide withdrawal and weight regain, Ly6Chi CCR2+ monocytes rebounded to levels seen in obese mice. These findings suggest that tirzepatide uncouples WL from the broad hematopoietic suppression seen in CR by preserving progenitor activity but selectively remodeling inflammatory/classical monocytes. We demonstrate that WL modality differentially impacts hematopoietic adaptation and provide evidence that classical monocytes are an effector cell through which tirzepatide may dampen obesity-associated inflammation.

ABSTRACT:T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy arising from the neoplastic transformation of immature T cells during their development in the thymus. Deciphering the developmental programs whose dysregulation drives T-ALL pathogenesis is critical for the development of novel targeted therapies, which remain an urgent unmet need for the treatment of this disease. MicroRNAs (miRNAs) have emerged as key posttranscriptional regulators of numerous physiological processes, including cancer. However, the specific role of miRNAs in human T-cell development and T-ALL pathogenesis remains largely unexplored. In this study, we comprehensively evaluated miRNA expression profiles across human T-cell development using microarray analysis and identified a dynamic expression pattern of miR-16-2, which is upregulated during early pre-T-cell proliferative stages up to the resting stage of immature thymocytes immediately preceding T-cell receptor αβ expression and is subsequently downregulated. We also confirmed the coordinated regulation of miR-15b expression, consistent with the reported clustered genomic location of both miRNAs. Notably, functional studies identified the miR-15b/16-2 cluster as a negative regulator of early thymocyte proliferation and demonstrated that overexpression of miR-15b/16-2 in T-ALL cells impaired leukemic growth in vitro and tumor progression in patient-derived xenotransplantation assays. Mechanistically, miR-15b/16-2 represses the expression of the genes encoding BCL-2 and cyclin D3, thereby promoting apoptosis and cell cycle dysregulation in T-ALL cells, characterized by an accumulation of G0-phase cells and a defective transition to the G2/M phase. Overall, these findings support a novel tumor-suppressive function for miR-15b/16-2 in T-ALL and highlight its potential as a promising therapeutic target.

Abstract Proper timing of DNA replication relies on sufficient nucleotide pools and replication machinery. The upstream regulatory programs that support the biomass production needed for DNA replication, particularly in the accelerated growth setting of cancer, remain incompletely defined. Here we show that the transcription factor ATF4 coordinates amino acid and nucleotide metabolism with selective protein synthesis to ensure proper DNA replication initiation and timing in acute leukemia. Specifically, ATF4 promotes the expression of enzymes that biosynthesize amino acids required for nucleotide production and drive the transcription of tRNA charging enzymes that sustain translation of a subset of proteins involved in replication origin firing. Consequently, ATF4 inhibition limits nucleotide biosynthesis and replication machinery, thereby disrupting DNA replication timing and leading to leukemia cell differentiation and death. Our findings indicate that ATF4 coordinates metabolic and translational programs to maintain DNA replication fidelity and the differentiation blockade in leukemia cells.

MicroRNAs (miRNAs) are small non-coding RNAs that play essential roles in gene regulation, cellular function, and disease pathogenesis. Advances in single-cell RNA sequencing technologies have enabled the profiling of miRNAs at single-cell resolution, providing unprecedented insight into cell-specific regulatory networks and heterogeneity. This chapter presents an overview of miRNA biology, technical approaches for single-cell miRNA sequencing, and recent bioinformatics tools developed for data analysis. We discuss challenges in library preparation, such as adapter biases and low RNA input, and highlight integrative strategies for co-profiling miRNAs with other omics layers. Finally, we outline in the conclusion the potential of single-cell miRNA profiling to contribute to precision medicine and therapeutic development, including its possible use in biomarker discovery, monitoring tumor heterogeneity, and informing personalized treatment strategies. As the field progresses, continued innovation will be critical to overcoming existing barriers and fully harnessing the power of single-cell miRNA analyses.

Introduction: Myelodysplastic syndromes (MDS) are hematologic malignancies characterized by bone marrow failure and risk for progression to acute myeloid leukemia (AML). Treatment for MDS depends on patients' risk of transformation to AML and death, calculated using the International Prognostic Scoring System-Revised (IPSS-R). For patients with higher-risk disease, the NCCN guidelines recommend combination therapy with a hypomethylating agent with or without venetoclax, among other targeted agents. Previous studies included a Phase 1b trial assessing combination therapy with azacitidine and venetoclax in adults with higher-risk MDS. Results included a complete remission rate of 30% and a median overall survival (OS) of 26 months. Furthermore, the Phase 3 VERONA trial did not meet the primary endpoint of OS with a hazard ratio of 0.908. The goal of the current study was to analyze outcomes in patients with higher-risk MDS treated with azacitidine and venetoclax at the Huntsman Cancer Institute. Methods: This is a single-center, retrospective study including patients with MDS who received at least one dose of azacitidine and venetoclax between January 1, 2022 and July 31, 2024. The primary outcome was overall response rate (ORR). Secondary outcomes included progression free survival (PFS), OS and time to best response. Safety outcomes included incidence of neutropenia, thrombocytopenia, neutropenic fever, sepsis and bacteremia. Results: Eighteen patients were included. The median age was 67-years-old (range 54-77), median bone marrow blast percentage was 12.2 (range 3-19), 66.7% of patients had not received prior treatment, 72.2% of patients had a TP53 mutation, 94.5% of patients had high or very high risk per IPSS-R. At diagnosis, 55.6% had MDS with biallelic TP53 inactivation and 33.3% had a myeloid neoplasm post cytotoxic therapy per WHO 2022. Azacitidine was administered at 75 mg/m2 IV daily for 5-7 days per cycle. Venetoclax dosing ranged from 100 mg to 400 mg daily adjusted for azole antifungal use. Duration of venetoclax varied with a median of 9 days of therapy across all cycles (range 6-21 days). Sixteen patients were analyzed for the primary outcome. The ORR was 81.3%, including 12 bone marrow complete responses (CR) and 1 partial response (PR). Of the CRs, 2 had unknown blood counts at the time of marrow assessment, and 4 met IWG 2006 but not IWG 2023 criteria due to the lack of count recovery. All CRs occurred within 1-2 cycles, with a median time to first response of 25 days. Four patients (22.2%) proceeded to allogeneic stem cell transplant (SCT). All patients (n = 18) were evaluated for survival outcomes. Median PFS was 4.2 months (95% CI 1.3 months-not reached) in the overall population, 3.7 months (95% CI 1-5.8 months) in patients with a TP53 mutation, and 17.3 months (95% CI 1.1 months-not reached) in patients without a TP53 mutation. Median OS was 5.6 months (95% CI 2.6-9.7 months) in the overall population, 4.4 months (95% CI 2.1-8.4 months) in patients with a TP53 mutation, and 17.3 months (95% CI 2.1 months-not reached) in patients without a TP53 mutation. Median time to transformation to AML was 4.2 months (95% CI 2.1 months-not reached). All patients developed Grade 3 or 4 neutropenia with a median time to absolute neutrophil count above 1000 x109 cells/L of 33 days. Fifteen patients (83.3%) developed grade 3-4 thrombocytopenia with a median time to platelet count above 50 x109 cells/L of 23.5 days. Ten patients (55.6%) developed neutropenic fever, and 7 patients (38.9%) developed bacteremia or sepsis. Discussion: In this population of high and very high risk MDS patients receiving azacitidine and venetoclax, the ORR was high at 81.3%. Due to high response rates, this regimen may be beneficial as a bridge for patients proceeding with allogeneic SCT. PFS and OS were considerably shorter in patients with a TP53 mutation indicating a lack of sustained benefit in this population. There are several limitations to this study including the retrospective nature of the analysis, variability in venetoclax dosing and duration, and inability to access outside records. Conclusion: Treatment with azacitidine and venetoclax in patients with higher-risk MDS may be helpful to achieve disease control quickly providing benefit in those intending to proceed to SCT. In our population, azacitidine and venetoclax did not provide long term disease control, especially in TP53-mutated disease.

High Mobility Group Box-1 (HMGB1) is a non-histone chromosome binding protein that has a dual function. Intracellular HMGB1 binds to DNA and is involved in transcriptional regulation, DNA replication, DNA repair, telomere maintenance, and nucleosome assembly. HMGB1 can also be passively released by necrotic or stressed cells, or actively secreted. When secreted, HMGB1 acts as a chemokine playing the role of a Damage-Associated Molecular Pattern (DAMP). As a DAMP, HMGB1 is involved in inflammatory response and signals through multiple receptors, including the Receptor for Advanced Glycation End Products (RAGE). Autophagy is a self-protective process that promotes cell survival under stress conditions. Autophagy degrades long-lived proteins, damaged organelles and abnormal protein aggregates. Autophagy induces cancer cell drug resistance, including in leukemia cells. When released by dying cancer cells, HMGB1 can signal through RAGE to induce autophagy in adjacent cells, potentially inducing resistance to chemotherapy. Extracellular HMGB1 was shown to interact with RAGE to induce autophagy and inhibit apoptosis in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia cells. This was associated with activation of ERK1/2 and decreased phosphorylation of mTOR. In addition, the anti-apoptotic effect of HMGB1 was associated with upregulation of bcl2, an effect that was RAGE-dependent. Azeliragon (AZE) is an orally available small molecule inhibitor of RAGE-ligand binding. It initially entered human clinical trials for the treatment of Alzheimer's disease where it was well tolerated. AZE is currently in clinical trials for the treatment of solid tumors. The goal of this study was to investigate the effect of AZE on AML cell lines (HL-60 and OCI-AML3) and patient-derived AML cells. HL-60 and OCI-AML3 were cultured in RPMI-1640 with 10% fetal bovine serum. Patient-derived cells were cultured in SFEM II, CD34 Expansion Supplement, and UM729. Patient-derived cells (4 from peripheral blood, 1 from bone marrow aspirate) were plated within 18 hours of collection and cultured for 1-3 days prior to addition of AZE. One additional patient-derived sample was collected by leukapheresis and cultured for 24 days to expand the rare blast population before adding AZE. Cells were incubated for 72 hours with 0-10 µM of AZE, before analysis by MTS (cell lines) or CellTiter Glo (patient cells). HL-60 and OCI-AML3 cell growth were completely eradicated by 3 µM AZE, with IC50 values of 2.2 µM and 1.7 µM, respectively. Synergy analysis of OCI-AML3 cells with Combenefit software demonstrated high synergy scores when AZE was combined with cytarabine or venetoclax, medium synergy scores when combined with daunorubicin, and antagonism when combined with azacitidine. HL60-cells showed no consistent synergy or antagonism signal across the three models in the Combenefit analysis with any drug combination. Blast-enriched samples from 6 AML patients (3 de novo untreated, 3 relapse) were incubated with 0-10 µM AZE. Cells were completely eradicated by 7.5-10 µM AZE (IC50=2-7 µM). The relapse patients included 1 with relapse after 2 bone marrow transplants, 1 with relapse after 7 lines of treatment (including Revumenib), and another 1 with relapse after Revumenib. AZE was effective against AML cells with KMT2A point mutations, duplications, gains, and rearrangements, suggesting a potential treatment option for patients after the use of menin inhibitors. Synergy analysis indicated synergy scores >30 with ≤5 µM AZE combined with 10 nM venetoclax and 0.75 µM azacitidine for 5/6 samples. The remaining sample was eradicated at 7.5 µM AZE, with a synergy score of 10 at 3.75 µM AZE. We conclude that AZE shows promising single agent activity in AML with potential for synergistic combinations with standard agents. Clinical investigation of AZE for the treatment of AML is warranted.

BACKGROUND:Decitabine efficacy in acute myeloid leukemia (AML) may be enhanced by the pharmacologic upregulation of microRNA miR-29b, a regulator of DNA methyltransferase (DNMT) expression. Bortezomib and sorafenib have been shown preclinically to increase miR-29b levels, providing a biologically informed strategy to sensitize leukemic blasts to DNMT inhibition. OBJECTIVES:To evaluate the safety, tolerability, biological activity, and preliminary efficacy of combining bortezomib and sorafenib followed by decitabine in patients with newly diagnosed or relapsed/refractory AML. METHODS:This phase I, dose-escalation study enrolled 15 patients (11 untreated, 4 relapsed/refractory) who received fixed-dose bortezomib and sorafenib across three dose levels prior to decitabine. Dose escalation was guided by dose-limiting toxicities (DLTs) and an increase in miR-29b expression. RESULTS:The regimen was generally well tolerated with the most frequent grade ≥3 adverse events of hypertension and febrile neutropenia. At the highest dose level, a ≥2-fold increase in miR-29b expression was observed in two of the six evaluable patients. The overall response rate was 33.3%, with clinical responses observed in both newly diagnosed and relapsed/refractory patients. However, changes in miR-29b expression did not consistently correlate with clinical response. CONCLUSIONS:Sequential treatment with bortezomib and sorafenib followed by decitabine is feasible and demonstrates acceptable safety in AML. Although the biologic modulation of miR-29b was variable, this trial provides a proof of concept for pharmacodynamic-guided dose finding in epigenetic therapy combinations.

Understanding the pathways regulating normal and malignant hematopoietic stem cell (HSC) biology is important for improving outcomes for patients with hematologic disorders. Epithelial Growth Factor Like-7 (EGFL7 ) is ∼30 kDa secreted protein that is highly expressed in adult HSCs. Using Egfl7 genetic knock-out ( Egfl7 KO) mice and recombinant EGFL7 (rEGFL7) protein, we examined the role of Egfl7 in regulating normal hematopoiesis. We found that Egfl7 KO mice had decreases in overall BM cellularity resulting in significant reduction in the number of hematopoietic stem and progenitor cells (HSPCs), which was due to dysregulation of normal cell-cycle progression along with a corresponding increase in quiescence. rEGFL7 treatment rescued our observed hematopoietic defects of Egfl7 KO mice and enhanced HSC expansion after genotoxic stress such as 5-FU and irradiation. Furthermore, treatment of WT mice with recombinant EGFL7 (rEGFL7) protein expands functional HSCs evidenced by an increase in transplantation potential. Overall, our data demonstrates a role for EGFL7 in HSC expansion and survival and represents a potential strategy for improving transplant engraftment or recovering bone marrow function after stress.

Limitations in the availability and expansion of hematopoietic stem cell (HSC) are a critical barrier for treating hematological disorders. Thus, understanding the regulatory mechanisms of HSC renewal, differentiation, and proliferation is crucial for developing techniques seeking to expand the HSC pool without compromising their multilineage potential. Epithelial Growth Factor Like-7 (EGFL7) is a ~30 kDa secreted protein highly expressed in adult bone marrow (BM) HSCs. Predicted gene expression profiling among tissues has revealed the highest level of protein expression in CD34+ hematopoietic cells (Hong et al., 2017); however, the precise role of EGFL7 in HSC regulation has not been thoroughly examined and remains poorly understood. To investigate the functional role of EGFL7 in HSC regulation, we used a germline Egfl7 knock-out (Egfl7 KO) mouse model. Egfl7 KO mice had 21% less BM cellularity and 36% lower numbers of long-term HSCs than WT controls (Lin-/Sca1+/cKit+/CD48-/CD150+/CD34-) (n=4-6, p<0.01 and p<0.05 respectively). To evaluate the function of EGFL7 in hematopoietic reconstitution, we performed competitive transplants of Egfl7 KO or WT sorted BM Lin-/Sca-1+/cKit+ (LSK) cells with WT LSK competitors (1:1, n=4-6). Egfl7 KO cells showed significantly reduced peripheral blood (PB) and BM engraftment 16 weeks post-transplant (32% vs 64%, 24% vs 45% respectively) and 19% less BM cellularity (p<0.05). We also performed serial competitive whole BM transplants of Egfl7 KO or WT with WT competitors (10:1 ratio, n=4-5) and found decreased reconstitution potential as evidenced by lower PB donor engraftment in the secondary transplants of Egfl7 KO group 38% vs 85%, p<0.001) as well as ~4-fold decrease in BM cellularity (p<0.05) and ~7-fold decrease in HSCs frequency (p<0.001), suggesting EGFL7 has a role in more primitive HSCs. To evaluate the role of EGFL7 in cell cycle regulation, we performed Bromodeoxyuridine (BrdU) incorporation in Egfl7 KO and WT mice and analyzed proliferating HSCs over 30 days while continuing BrdU treatment orally through water. Proliferating HSCs were lower in Egfl7 KO mice as early as day 3 (2.7% vs 4.8%, p<0.01). Furthermore, staining with Ki67 and DNA counterstain showed higher frequency of Egfl7 KO HSCs present in G0 phase (~84% vs ~72%, p<0.01), suggesting that EGFL7 is required for HSC activation and exit from quiescence. Next, we hypothesized that exogenous treatment with recombinant EGFL7 (rEGFL7) could expand HSCs in vivo and rescue the Egfl7 KO phenotype. We first treated WT mice with 10µg of rEGFL7 or vehicle daily for 10 days (n=3-6). rEGFL7-treated mice had 32% more HSCs (p<0.05), which led to an 18% increase in engrafting ability upon BM transplants (p<0.001). We then treated Egfl7 KO mice with rEGFL7; remarkably, treatment not only restored HSC numbers and BM cellularity, but also induced HSCs to leave quiescence and enter cell cycle, evidenced by ~2-fold decrease in G0 frequency and ~1.6-fold increase in proliferating HSCs (p<0.01). Taken together, these data show that EGFL7 enhances HSC functionality and is required for HSC proliferation. To examine molecular pathways regulated by EGFL7, we performed single-cell RNA sequencing (sc-RNA seq) on BM cKit+ cells from Egfl7 KO and WT mice. Consistent with our data, Egfl7 KO HSCsshowed higher percentage of cells in G1 (64% vs 54%) along with a reduction in their cell cycle score (-0.043 vs -0.035, p<0.05). Further analysis revealed overexpression of genes that regulate quiescence (Egr1, Btg2, Egf3, ler3), self-renewal (Klf2, Klf4, Klf6), and apoptotic markers (AP-1) (Fosb, Fos, Jun, Jund) in Egfl7 KO HSC. Given the potential of AP-1 factors to suppress cell cycle activity, we investigated phosphorylation levels of c-Fos by flow cytometry. We found higher levels of c-Fos phosphorylation (Ser32) in Egfl7 KO HSC, while exogenous rEGFL7 treatment reduced c-Fos phosphorylation levels in WT HSCs by ~45% (n=3, p<0.0001). AP-1 inhibition increased cycle progression in Egfl7 KO progenitor cells, as indicated by in vitro BrdU incorporation assay, suggesting that EGFL7 suppresses AP-1/c-Fos activity resulting in activation of quiescent HSCs. Our data uncovers the role of EGFL7 in the regulation of HSC cycle through inhibition of c-Fos to exit quiescence and subsequent entry into the cell cycle. Our findings provide a foundation for evaluating the potential therapeutic use of EGFL7 protein to expand the HSC pool while retaining functionality.

Circular RNAs are a novel class of RNA transcripts, which regulate important cellular functions in health and disease. Herein, we report on the functional relevance of the circPCMTD1 transcript in acute leukemias. In screening experiments, we found that circPCMTD1 depletion strongly inhibited the proliferative capacity of leukemic cells with BCR-ABL translocations. Mass cytometry experiments identified the aberrant activation of the DNA damage response as an early downstream event of circPCMTD1 depletion. In in vivo experiments, circPCMTD1 targeting prolonged the survival of mice engrafted with leukemic blasts harboring the Philadelphia chromosome. Mechanistically, we found that circPCMTD1 was enriched in the cytoplasm and associated with the ribosomes of the leukemic cells. We detected a cryptic open reading frame within the circPCMTD1 sequence and found that circPCMTD1 could generate a peptide product. The circPCMTD 1-derived peptide interacted with proteins of the BTR complex and enhanced BTR complex formation, thereby increasing tolerance to genotoxic stress.

Acute graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic cell transplantation (allo-HCT). Using preclinical mouse models of disease, previous work in our laboratory has linked microRNA-155 (miR-155) to the development of acute GVHD. Transplantation of donor T cells from miR-155 host gene (MIR155HG) knockout mice prevented acute GVHD in multiple murine models of disease while maintaining critical graft-versus-leukemia (GVL) response, necessary for relapse prevention. In this study, we used clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 genome editing to delete miR-155 in primary T cells (MIR155HG degrees exon3) from human donors, resulting in stable and sustained reduction in expression of miR-155. Using the xenogeneic model of acute GVHD, we show that NOD/SCID/IL2rynull (NSG) mice receiving MIR155HG degrees exon3 human T cells provide protection from lethal acute GVHD compared with mice that received human T cells with intact miR-155. MIR155HG degrees exon3 human T cells persist in the recipients displaying decreased proliferation potential, reduced pathogenic T helper-1 cell population, and infiltration into GVHD target organs, such as the liver and skin. Importantly, MIR155HG degrees exon3 human T cells retain GVL response significantly improving survival in an in vivo model of xeno-GVL. Altogether, we show that CRISPR/Cas9- mediated deletion of MIR155HG in primary human donor T cells is an innovative approach to generate allogeneic donor T cells that provide protection from lethal GVHD while maintaining robust antileukemic response.

Circular RNAs (circRNAs) are a novel class of RNA transcripts, which regulate important cellular functions in health and disease. CircRNAs are covalently joined and characterized by the perturbed arrangement of exons known as back-splicing. Initially regarded as transcriptional byproducts, circRNAs have been shown to regulate mRNA translation by acting as microRNA sponges, and recent studies have revealed their roles in transcription, translation, and various cellular functions. In cancer, circRNAs can function as oncogenes or tumor suppressors, and their stability makes them potential biomarkers for disease. In acute leukemias, circRNAs generated from recurrent chromosomal translocations contribute to leukemogenesis. Here, we investigate the role of circPCMTD1 in chronic myeloid leukemia (CML) in the blast crisis (BC). Functional studies using LNA-modified, RNase H-recruiting oligonucleotides (gapmers) targeting circPCMTD1 demonstrated a significant decrease in proliferation and a potent G2/M cell cycle blockade in CML-BC cell lines (K-562 & LAMA-84), both harboring the t(9;22)(q34;q11.2). Quantitative real-time PCR confirmed the specificity of circPCMTD1 depletion without affecting the linear PCMTD1 transcript. CircPCMTD1 knockdown (KD) reduced the viability of leukemic blasts, indicating its essential role in cell survival. RNA sequencing after circPCMTD1-KD in K-562 cells identified approximately 150 differentially expressed genes involved in cell cycle control, nuclear organization, and transcriptional regulation, such as SMARCA4, MACM, PCLAF, and SASH1. Gene Set Enrichment analysis highlighted rRNA processing and DNA replication-dependent chromatin function to be notably affected by circPCMTD1 depletion. CyTOF-based cell cycle analysis validated the G2/M blockade. Increased γH2AX levels indicated aberrant DNA damage response, confirmed by western blotting and intracellular flow cytometry. In addition, circPCMTD1-KD led to an increase in the phosphorylation of the CHK1, RPA32, ATR, ATM, and DNA-PK proteins. DNA fiber assays and comet assays further confirmed reduced DNA replication capacity and increased double-stranded DNA breaks upon circPCMTD1 depletion. Taken together, these data underscore the aberrant DNA damage response and the significant increase in genotoxic stress that is triggered by circPCMTD1 depletion. We performed targeted circPCMTD1 profiling in CML patients in the chronic, accelerated, and blast crisis phase and found an increased abundance of circPCMTD1 in advanced disease stages, indicating a potential role of higher circPCMTD1 expression in disease progression. In vitro experiments with patient blasts showed that circPCMTD1-KD increased γH2AX levels specifically in BCR::ABL-positive samples. In vivo, targeting circPCMTD1 in mice engrafted with BCR::ABL-positive blasts prolonged survival significantly, with no notable toxicities observed. Mechanistically, circPCMTD1 was enriched in the cytoplasm and associated with ribosomes. Polysome profiling suggested its protein-coding capacity, and we identified a cryptic open reading frame within circPCMTD1. Using custom antibodies, we detected a circPCMTD1-derived peptide (~30 KD) localized mainly in the nucleus. Immunoprecipitation followed by mass spectrometry revealed that the peptide interacted with BLM, TOP3A, and RMI1 proteins of the BTR complex. CircPCMTD1 knockdown reduced BTR complex formation. Knockdown of these proteins individually reduced leukemic blast viability, but concomitant depletion mimicked the G2/M blockade seen with circPCMTD1 depletion. Furthermore, treatment with Dasatinib, a tyrosine kinase inhibitor, decreased circPCMTD1-derived peptide levels without affecting the expression levels of the circPCMTD1 transcript and reduced BTR complex formation, linking BCR::ABL activity to circPCMTD1 function. In summary, we identify circPCMTD1 as a crucial regulator in BCR::ABL-positive leukemias, affecting DNA damage response, proliferation, and cell cycle progression. Our findings highlight circPCMTD1 as a potential therapeutic target in myeloid malignancies with t(9;22). Future studies should explore the therapeutic implications of targeting circPCMTD1 in combination with existing treatments, potentially offering a novel approach to managing drug resistance and improving outcomes in CML patients.

Leukemias arise from recurrent clonal mutations in hematopoietic stem/progenitor cells (HSPCs) that cause profound changes in the bone marrow microenvironment (BMM) favoring leukemic stem cell (LSC) growth over normal HSPCs. Understanding the cross talk between preleukemic mutated HSPCs and the BMM is critical to develop novel therapeutic strategies to prevent leukemogenesis. We hypothesize that preleukemic-LSCs (pLSCs) induce BMM changes critical for leukemogenesis. Using our AML-murine model, we performed single-cell RNA-sequencing of preleukemic BMM (pBMM) cells. We found normal HSC (nHSC)-regulating LepR+ mesenchymal stem cells, and endothelial cells were decreased, along with increases in CD55+ fibroblasts and pericytes. Preleukemic CD55+ fibroblasts had higher proliferation rates and decreased collagen expression, suggesting extracellular matrix remodeling during leukemogenesis. Importantly, co-culture assays found preleukemic CD55+ fibroblasts expanded pLSCs significantly over nHSCs. In conclusion, we have identified a distinct pBMM and a novel CD55+ fibroblast population that is expanded in pBMM that promote fitness of pLSCs over nHSCs.

Leukemias arise from recurrent clonal mutations in hematopoietic stem/progenitor cells (HSPCs) that cause profound changes in the bone marrow microenvironment (BMM) favoring leukemic stem cell (LSC) growth over normal HSPCs. Understanding the cross talk between preleukemic mutated HSPCs and the BMM is critical to develop novel therapeutic strategies to prevent/treat leukemogenesis. We hypothesize that preleukemic-LSCs (pLSCs) induce BMM changes critical for leukemogenesis. Using our AML-murine model, we performed single-cell RNA-sequencing of preleukemic BMM (pBMM) cells. We found that the pBMM is distinct from the normal BMM with decreases in mesenchymal stem cells (MSCs) and endothelial cells (ECs), with a concomitant increase in CD55+ fibroblasts and pericytes. Upon further characterization of these preleukemic CD55+ fibroblasts, we identified an increase in inflammatory cancer-associated fibroblasts (iCAFs) compared to myofibroblast CAFs (myCAFs). Preleukemic iCAFs had higher proliferation rates and decreased collagen expression along with increases in pro-inflammatory cytokines such as RANTES, IL-6, and IL-11. Importantly, preleukemic fibroblasts also preferentially expanded pLSCs over nHSCs in co-culture assays. In conclusion, we have identified a unique pBMM that is characterized by increases in iCAFs and promotes expansion of pLSCs.

It has been reported that elevated levels of miR-155 have been reported in patients with Acute Myeloid Leukemia (AML) bearing FLT3 -ITD mutations and is independent of FLT3 -ITD signaling. However, it is unclear how miR-155 is expressed in leukemic stem cells (LCS) and whether miR-155 has any role regulating LSC functions. In this manuscript, we defined the expression of miR-155 in clearly defined LCSs population and showed that miR-155 is regulating self-renewal and quiescence of LSCs.### Competing Interest StatementThe authors have declared no competing interest.

Understanding the pathways regulating normal and malignant hematopoietic stem cell (HSC) biology is important for improving outcomes for patients with hematologic disorders. Epithelial Growth Factor Like-7 (EGFL7) is ∼30 KDa secreted protein that is highly expressed in adult HSCs. Using Egfl7 genetic knock-out (Egfl7 KO) mice and recombinant EGFL7 (rEGFL7) protein, we examined the role of Egfl7 in regulating normal hematopoiesis. Egfl7 KO mice had decreased overall BM cellularity (Egfl7 KO – 1.20X10^8 BM cells/mouse vs WT - 1.51X10^8 BM cells/mouse; p< 0.01) and significant reduction in the number of hematopoietic stem and progenitor cells (HSP Cs) (Egfl7 KO – 4.52X10^5 LSK cells/mouse vs WT – 3.2X10^5 LSK cells/mouse; p< 0.05). We found this reduction in Eglf7 KO HSPCs was due to dysregulation of the cell-cycle resulting in increased HSC quiescence (Egfl7 KO – 38% vs WT – 14%; p< 0.01). Single-cell RNA sequencing revealed an increase in expression of quiescence-inducing molecule Egr1, supporting a role for EGFL7 in regulating HSC quiescence. Wild-type mice treated with recombinant rEGFL7 protein expanded the number of functional HSCs (control WT – 2X10^4 HSCs/mouse vs rEGFL7 treated WT – 3.1X10^4 HSCs/mouse; p< 0.05) resulting in increased donor chimerism in bone marrow transplant (BMT) assays. rEGFL7 treatment also rescued HSPC numbers in Egfl7 KO mice as a result of increased proliferation and survival of HSCs. Altogether, our data demonstrate an important role for EGFL7 in regulating HSCs and the potential use of rEGFL7 to expand functional HSCs for use in emerging cellular therapies.

Abstract Preleukemic stem cells (LSCs) can remain dormant within the bone marrow (BM) for decades before disease onset, suggesting acquisition of mutations within hematopoietic stem/progenitor cells (HSPCs) alone is insufficient for acute myeloid leukemia (AML). It is likely that alterations within BM microenvironment (BMM) also occur due to dysregulated crosstalk with mutant HSCs thus favoring LSC growth over normal HSCs. Uncovering novel mechanisms by which preleukemic LSCs remodel the BMM is critical in understanding therapy resistance and to develop novel therapies targeting aberrant pathways in both the LSCs and cells within the BMM. To address this, we compared cells of the BMM of wildtype (WT) mice to our Mll-PTD;Flt3-ITD mouse model of AML, which has a distinct preleukemic phase. Using single cell RNA-sequencing we identified 17 stromal cell clusters and observed substantial changes in both cell numbers and transcriptional profiles of several key stromal cell subpopulations. Specifically, we found decreases in normal HSC regulatory populations such as LepR+ MSCs and Cdh5+ ECs (2 and 2.7-fold decreases, respectively) and increases in pericytes and fibroblasts in preleukemic BM compared to WT (2.7 and 3.5-fold increase, respectively). Unlike what is observed in full-blown AML, we found an increase in CD55+ fibroblasts resulting from increased proliferation (13% vs 6% cell cycling, P=0.0016). Importantly, co-culture assays found that CD55+ BM fibroblasts expanded LSCs at increased levels over normal HSCs (83+/-7.5 vs 57+/-10.1, P=0.0006). Downregulation of several collagen genes (Col1a1, Col1a2, Col3a1, Col4a1, and Col6a1, P< 0.05) was found in these preleukemic CD55+ fibroblasts, suggesting that remodeling of the extracellular matrix might be an important mechanism by which these CD55+ fibroblast remodel the normal BMM and promote leukemogenesis. Furthermore, sequencing of full-blown leukemic BM niche cells have identified several key differences in the BM architecture, suggesting that the preleukemic BM is indeed distinct from full-blown leukemic BM and is important for disease initiation and progression, and not disease maintenance. Citation Format: Chinmayee Goda, Rohan Kulkarni, Alexander Rudich, Malith Karunasiri, Yaphet Bustos, Sadie Chidester, Ozlen Balcioglu, Elizabeth AR Garfinkle, Bethany Mundy-Bosse, Elaine R Mardis, Guido Marcucci, Ramiro Garzon, Katherine Miller, Adrienne M Dorrance. Identification of the unique preleukemic bone marrow niche in acute myeloid leukemia [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P07.

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