Expansion Of Hematopoietic Stem Cells Research Articles

Loss of IDH1 and IDH2 in macrophages induces pathological phenotypes in cardiomyocytes

Abstract Introduction The clonal expansion of hematopoietic stem cells can be induced by a variety of age-related somatic mutations, a phenomenon known as "clonal hematopoiesis of indeterminate potential" (CHIP). CHIP is associated with an increased incidence and poor outcomes in patients with cardiovascular disease. The mechanisms underlying the crosstalk between CHIP cells and cardiac tissue are yet to be elucidated. Rare mutations in the metabolism-regulating isocitrate dehydrogenases encoding genes IDH1 and IDH2 have been identified as AML-like and cardiovascular high-risk CHIP-driving mutations in patient cohorts. However, the potential paracrine effects of these mutations in immune cells on cardiac tissue are unknown. Therefore, we assessed the impact of IDH1 and IDH2 mutations on the paracrine activities of macrophages. Methods and Results To gain first insights into the functional consequences of the IDH mutations, we used the AlphaMissense algorithm, which predicts the impact of mutations on protein function. IDH1 and IDH2 mutations identified in our patient cohorts were predicted to induce pathological alterations of protein function with a score >0.99. Therefore, we assessed the effects of silencing of the two genes in primary human CD14+ M0 macrophages on the paracrine activity on neonatal rat cardiomyocytes. Transfection with siRNA pools targeting IDH1 and IDH2 to mimic CHIP-driving conditions reduced mRNA abundance by 78% and 73%, respectively (both p<0.05). To assess the impact of IDH-silencing on cardiovascular cells, cardiomyocytes were treated with the supernatant of the macrophages. Interestingly, supernatants of IDH1 silenced macrophages significantly increased apoptosis of cardiomyocytes (8.6-fold increased expression of cleaved caspase-3; p<0.05) and reduced beating frequency. In addition, supernatants of IDH1 silenced macrophages induced a 1.5-fold increase in cardiomyocyte hypertrophy (p<0.01), whereas silencing of IDH2 showed no effect on cardiomyocyte hypertrophy and apoptosis. However, supernatants of both IDH1 and IDH2 silenced macrophages resulted in significantly increased numbers of bi- and multinucleated cardiomyocytes. Conclusion: Loss of isocitrate dehydrogenases in human macrophages affects cardiomyocyte functions in a paracrine fashion. Particularly silencing of IDH1 in macrophages introduced cardiomyocyte hypertrophy, apoptosis, and bi- and multinucleation, and reduced contractile function. Ongoing studies will address how IDH1 and IDH2 silencing-induced alterations in macrophages control their paracrine activity on cardiomyocytes.

Genomic instability and genetic heterogeneity in aging: insights from clonal hematopoiesis (CHIP), monoclonal gammopathy (MGUS), and monoclonal B-cell lymphocytosis (MBL).

Aging is a multifaceted process characterized by a gradual decline in physiological function and increased susceptibility to a range of chronic diseases. Among the molecular and cellular mechanisms driving aging, genomic instability is a fundamental hallmark, contributing to increased mutation load and genetic heterogeneity within cellular populations. This review explores the role of genomic instability and genetic heterogeneity in aging in the hematopoietic system, with a particular focus on clonal hematopoiesis of indeterminate potential (CHIP), monoclonal gammopathy of undetermined significance (MGUS), and monoclonal B-cell lymphocytosis (MBL) as biomarkers. CHIP involves the clonal expansion of hematopoietic stem cells with somatic mutations. In contrast, MGUS is characterized by the presence of clonal plasma cells producing monoclonal immunoglobulins, while MBL is characterized by clonal proliferation of B cells. These conditions are prevalent in the aging population and serve as measurable indicators of underlying genomic instability. Studying these entities offers valuable insights into the mechanisms by which somatic mutations accumulate and drive clonal evolution in the hematopoietic system, providing a deeper understanding of how aging impacts cellular and tissue homeostasis. In summary, the hematopoietic system serves as a powerful model for investigating the interplay between genomic instability and aging. Incorporating age-related hematological conditions into aging research, alongside other biomarkers such as epigenetic clocks, can enhance the precision and predictive power of biological age assessments. These biomarkers provide a comprehensive view of the aging process, facilitating the early detection of age-related diseases and hopefully enabling personalized healthcare strategies.

Myeloid deficiency of heparan sulfate 6-O-endosulfatases impairs bone marrow hematopoiesis

The heparan sulfate (HS) 6-O-endosulfatases or the Sulfs (Sulf1 and Sulf2) are the only known enzymes that can modify HS sulfation status extracellularly and have been shown to regulate diverse biological processes. The role of the Sulfs in bone marrow (BM) hematopoiesis is not known. In this study, we generated a novel mouse line with myeloid-specific deletion of the Sulfs by crossing Sulf1/2 double floxed mice with the LysM-cre line. The LysM-Sulf knockout (KO) male mice exhibited age-dependent expansion of hematopoietic stem cells and the granulocyte-monocyte lineages in the BM, whereas common lymphoid progenitors and B lymphocyte populations were significantly reduced. Although megakaryocytic and erythroid progenitors were not reduced in the BM, the LysM-Sulf KO males suffered age-dependent reduction of red blood cells (RBCs) and platelets in the peripheral blood, suggesting that the production of RBCs and platelets was arrested at later stages. In addition, LysM-Sulf KO males displayed progressive splenomegaly with extramedullary hematopoiesis. Compared to males, LysM-Sulf KO females exhibited a much-reduced phenotype, and ovariectomy had little effect. Mechanistically, reduced TGF-β/Smad2 but enhanced p53/p21 signaling were observed in male but not female LysM-Sulf KO mice. Finally, HS disaccharide analysis via LC-MS/MS revealed increased HS 6-O-sulfation in the BM from both male and female LysM-Sulf KO mice, however, the distribution of 6-O-sulfated motifs were different between the sexes with compensatory increase in Sulf1 expression observed only in LysM-Sulf KO females. In conclusion, our study reveals that myeloid deficiency of the Sulfs leads to multilineage abnormalities in BM hematopoiesis in an age- and sex-dependent manner.

The evolving hematopoietic niche during development.

Mammalian hematopoietic stem cells (HSCs) emerge from the hemogenic endothelium in the major embryonic arteries. HSCs undergo a complex journey first migrating to the fetal liver (FL) and from there to the fetal bone marrow (FBM), where they mostly remain during adult life. In this process, a pool of adult HSCs is produced, which sustains lifelong hematopoiesis. Multiple cellular components support HSC maturation and expansion and modulate their response to environmental and developmental cues. While the adult HSC niche has been extensively studied over the last two decades, the niches present in the major embryonic arteries, FL, FBM and perinatal bone marrow (BM) are poorly described. Recent investigations highlight important differences among FL, FBM and adult BM niches and emphasize the important role that inflammation, microbiota and hormonal factors play regulating HSCs and their niches. We provide a review on our current understanding of these important cellular microenvironments across ontogeny. We mainly focused on mice, as the most widely used research model, and, when possible, include relevant insights from other vertebrates including birds, zebrafish, and human. Developing a comprehensive picture on these processes is critical to understand the earliest origins of childhood leukemia and to achieve multiple goals in regenerative medicine, such as mimicking HSC development in vitro to produce HSCs for broad transplantation purposes in leukemia, following chemotherapy, bone marrow failure, and in HSC-based gene therapy.

Clonal hematopoiesis and hematological malignancy.

Clonal hematopoiesis (CH), the expansion of hematopoietic stem cells and their progeny driven by somatic mutations in leukemia-associated genes, is a common phenomenon that rises in prevalence with advancing age to affect most people older than 70 years. CH remains subclinical in most carriers, but, in a minority, it progresses to a myeloid neoplasm, such as acute myeloid leukemia, myelodysplastic syndrome, or myeloproliferative neoplasm. Over the last decade, advances in our understanding of CH, its molecular landscape, and the risks associated with different driver gene mutations have culminated in recent developments that allow for a more precise estimation of myeloid neoplasia risk in CH carriers. In turn, this is leading to the development of translational and clinical programs to intercept and prevent CH from developing into myeloid neoplasia. Here, we give an overview of the spectrum of CH driver mutations, what is known about their pathophysiology, and how this informs the risk of incident myeloid malignancy.

Further biological characterization of small molecules UM171 and SR1: In vitro effects on three hematopoietic cell populations from human cord blood

Small molecules UM171 and SR1 have already been taken into clinically-oriented protocols for the ex vivo expansion of hematopoietic stem (HSCs) and progenitor (HPCs) cells. In order to gain further insight into their biology, in the present study we have assessed their effects, both individually and in combination, on the in vitro long-term proliferation and expansion of HSCs and HPCs contained within three different cord blood-derived cell populations: MNCs (CD34+ cells = 0.8 %), LIN− cells (CD34+ cells = 41 %), and CD34+ cells (CD34+ cells >98 %). Our results show that when added to cultures in the absence of recombinant stimulatory cytokines, neither molecule had any effect. In contrast, when added in the presence of hematopoietic cytokines, UM171 and SR1 had significant stimulatory effects on cell proliferation and expansion in cultures of LIN− and CD34+ cells. No significant effects were observed in cultures of MNCs. The effects of both molecules were more pronounced in cultures with the highest proportion of CD34+ cells, and the greatest effects were observed when both molecules were added in combination. In the absence of small molecules, cell numbers reached a peak by days 25–30, and then declined; whereas in the presence of UM171 or/and SR1 cell numbers were sustained up to day 45 of culture. Our results indicate that besides CD34+ cells, LIN− cells could also be used as input cells in clinically-oriented expansion protocols, and that using both molecules simultaneously would be a better approach than using only one of them.

MTORC1 mediates the expansion of hematopoietic stem and progenitor cells through ribosome biogenesis protein Urb2 in zebrafish

Mammalian target of rapamycin (mTOR) serves as the key sensor to control protein synthesis, cell growth, and survival. Despite mTOR is reported to regulate hematopoietic stem and progenitor cell (HSPC) engraftment and multiple-lineage hematopoiesis in mice, the roles of unique mTOR complexes (mTORCs) in early HSPC development and HSPC pool formation have not been adequately elucidated. Here, we uncover that mTORC1 is essential for early HSPC expansion in zebrafish. mTORC1 signaling was highly activated in definitive HSPCs during the emerging and expanding stages. Pharmacological or genetic inactivation of mTORC1 would cause defective HSPC expansion and migration due to disrupted cell proliferation. Interestingly, mTORC2 is dispensable for early HSPC development. Ribosome biogenesis protein Urb2 was downregulated upon mTORC1 inhibition, and urb2 overexpression partially rescued the hematopoietic defects in mTORC1-deficient embryos. These data demonstrate that mTORC1 signaling regulates early HSPC expansion through Urb2, and this work will deepen our understanding of mTOR in different physiological processes.

CHIP: a clonal odyssey of the bone marrow niche.

Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by the selective expansion of hematopoietic stem and progenitor cells (HSPCs) carrying somatic mutations. While CHIP is typically asymptomatic, it has garnered substantial attention due to its association with the pathogenesis of multiple disease conditions, including cardiovascular disease (CVD) and hematological malignancies. In this Review, we will discuss seminal and recent studies that have advanced our understanding of mechanisms that drive selection for mutant HSPCs in the BM niche. Next, we will address recent studies evaluating potential relationships between the clonal dynamics of CHIP and hematopoietic development across the lifespan. Next, we will examine the roles of systemic factors that can influence hematopoietic stem cell (HSC) fitness, including inflammation, and exposures to cytotoxic agents in driving selection for CHIP clones. Furthermore, we will consider how - through their impact on the BM niche - lifestyle factors, including diet, exercise, and psychosocial stressors, might contribute to the process of somatic evolution in the BM that culminates in CHIP. Finally, we will review the role of old age as a major driver of selection in CHIP.

Isolation of Human Hematopoietic Stem Cells from an Apheresis Sample.

The hematopoietic system constantly produces new blood cells through hematopoiesis, and maintaining this balance is vital for human health. This balance is maintained by self-renewing hematopoietic stem cells (HSCs) and various progenitor cells. Under typical circumstances, HSCs are not abundantly found in peripheral blood; hence, their mobilization from the bone marrow is vital. Hematopoietic growth factors achieve this effectively, enabling mobilization and thus allowing blood sample and thus HSC collection via apheresis. Securing a sufficient supply of HSCs is vital for successful hematopoietic reconstitution and the rapid integration of committed cells. Thus, isolation and expansion of HSCs are crucial for convenient extraction, production of transplantable quantities, genetic modifications for enhanced therapeutic efficacy, and as a source of increased/expanded/synthesized blood cells in vitro. In conclusion, the isolation and expansion of HSCs play pivotal roles in both regenerative medicine and hematology. This protocol describes the isolation of human HSCs by providing an overview of the primary method for isolating human hematopoietic stem cells from apheresis blood samples and sheds light on human HSC studies and developments in research and medicine.

Exploring the potential of predicted miRNAs on the genes involved in the expansion of hematopoietic stem cells

A major challenge in therapeutic approaches applying hematopoietic stem cells (HSCs) is the cell quantity. The primary objective of this study was to predict the miRNAs and anti-miRNAs using bioinformatics tools and investigate their effects on the expression levels of key genes predicted in the improvement of proliferation, and the inhibition of differentiation in HSCs isolated from Human umbilical cord blood (HUCB). A network including genes related to the differentiation and proliferation stages of HSCs was constructed by enriching data of text (PubMed) and StemChecker server with KEGG signaling pathways, and was improved using GEO datasets. Bioinformatics tools predicted a profile from miRNAs containing miR-20a-5p, miR-423-5p, and chimeric anti-miRNA constructed from 5′-miR-340/3′-miR-524 for the high-score genes (RB1, SMAD4, STAT1, CALML4, GNG13, and CDKN1A/CDKN1B genes) in the network. The miRNAs and anti-miRNA were transferred into HSCs using polyethylenimine (PEI). The gene expression levels were estimated using the RT-qPCR technique in the PEI + (miRNA/anti-miRNA)-contained cell groups (n = 6). Furthermore, CD markers (90, 16, and 45) were evaluated using flow cytometry. Strong relationships were found between the high-score genes, miRNAs, and chimeric anti-miRNA. The RB1, SMAD4, and STAT1 gene expression levels were decreased by miR-20a-5p (P < 0.05). Additionally, the anti-miRNA increased the gene expression level of GNG13 (P < 0.05), whereas the miR-423-5p decreased the CDKN1A gene expression level (P < 0.01). The cellular count also increased significantly (P < 0.05) but the CD45 differentiation marker did not change in the cell groups. The study revealed the predicted miRNA/anti-miRNA profile expands HSCs isolated from HUCB. While miR-20a-5p suppressed the RB1, SMAD4, and STAT1 genes involved in cellular differentiation, the anti-miRNA promoted the GNG13 gene related to the proliferation process. Notably, the mixed miRNA/anti-miRNA group exhibited the highest cellular expansion. This approach could hold promise for enhancing the cell quantity in HSC therapy.

Effect of Post-transplant Dietary Restriction on Hematopoietic Reconstitution and Maintenance of Reconstitution Capacity of Hematopoietic Stem Cells.

Hematopoietic cell transplantation (HCT) is an important therapy for many hematological malignancies as well as some non-malignant diseases. Post-transplant hematopoiesis is affected by multiple factors, and the mechanisms of delayed post-transplant hematopoiesis remain poorly understood. Patients undergoing HCT often suffer from significantly reduced food intake due to complications induced by preconditioning treatments. Here, we used a dietary restriction (DR) mouse model to study the effect of post-transplant dietary reduction on hematopoiesis and hematopoietic stem cells (HSCs). We found that post-transplant DR significantly inhibited both lymphopoiesis and myelopoiesis in the primary recipient mice. However, when bone marrow cells (BMCs) from the primary recipient mice were serially transplanted into secondary and tertiary recipient mice, the HSCs derived from the primary recipient mice, which were exposed to post-transplant DR, exhibited a much higher reconstitution capacity. Transplantation experiments with purified HSCs showed that post-transplant DR greatly inhibited hematopoietic stem cell (HSC) expansion. Additionally, post-transplant DR reshaped the gut microbiotas of the recipient mice, which inhibited inflammatory responses and thus may have contributed to maintaining HSC function. Our findings may have important implications for clinical work because reduced food intake and problems with digestion and absorption are common in patients undergoing HCT.

Genetic deletion of JAM-C in preleukemic cells rewires leukemic stem cell gene expression program in AML

AbstractThe leukemic stem cell (LSC) score LSC-17 based on a stemness–related gene expression signature is an indicator of poor disease outcome in acute myeloid leukemia (AML). However, it is not known whether “niche anchoring” of LSC affects disease evolution. To address this issue, we conditionally inactivated the adhesion molecule JAM-C (Junctional Adhesion Molecule-C) expressed by hematopoietic stem cells (HSCs) and LSCs in an inducible mixed-lineage leukemia (iMLL)-AF9–driven AML mouse model. Deletion of Jam3 (encoding JAM-C) before induction of the leukemia–initiating iMLL-AF9 fusion resulted in a shift from long-term to short-term HSC expansion, without affecting disease initiation and progression. In vitro experiments showed that JAM-C controlled leukemic cell nesting irrespective of the bone marrow stromal cells used. RNA sequencing performed on leukemic HSCs isolated from diseased mice revealed that genes upregulated in Jam3-deficient animals belonged to activation protein-1 (AP-1) and tumor necrosis factor α (TNF-α)/NF-κB pathways. Human orthologs of dysregulated genes allowed to identify a score that was distinct from, and complementary to, the LSC-17 score. Substratification of patients with AML using LSC-17 and AP-1/TNF-α genes signature defined 4 groups with median survival ranging from <1 year to a median of “not reached” after 8 years. Finally, coculture experiments showed that AP-1 activation in leukemic cells was dependent on the nature of stromal cells. Altogether, our results identify the AP-1/TNF-α gene signature as a proxy of LSC anchoring in bone marrow niches, which improves the prognostic value of the LSC-17 score. This trial was registered at www.ClinicalTrials.gov as #NCT02320656.

Chronic TNF in the aging microenvironment exacerbates Tet2 loss-of-function myeloid expansion

AbstractSomatic mutations in the TET2 gene occur more frequently with age, imparting an intrinsic hematopoietic stem cells (HSCs) advantage and contributing to a phenomenon termed clonal hematopoiesis of indeterminate potential (CHIP). Individuals with TET2-mutant CHIP have a higher risk of developing myeloid neoplasms and other aging-related conditions. Despite its role in unhealthy aging, the extrinsic mechanisms driving TET2-mutant CHIP clonal expansion remain unclear. We previously showed an environment containing tumor necrosis factor (TNF) favors TET2-mutant HSC expansion in vitro. We therefore postulated that age-related increases in TNF also provide an advantage to HSCs with TET2 mutations in vivo. To test this hypothesis, we generated mixed bone marrow chimeric mice of old wild-type (WT) and TNF–/– genotypes reconstituted with WT CD45.1+ and Tet2–/– CD45.2+ HSCs. We show that age-associated increases in TNF dramatically increased the expansion of Tet2–/– cells in old WT recipient mice, with strong skewing toward the myeloid lineage. This aberrant myelomonocytic advantage was mitigated in old TNF–/– recipient mice, suggesting that TNF signaling is essential for the expansion Tet2-mutant myeloid clones. Examination of human patients with rheumatoid arthritis with clonal hematopoiesis revealed that hematopoietic cells carrying certain mutations, including in TET2, may be sensitive to reduced TNF bioactivity following blockade with adalimumab. This suggests that targeting TNF may reduce the burden of some forms of CHIP. To our knowledge, this is the first evidence to demonstrate that TNF has a causal role in driving TET2-mutant CHIP in vivo. These findings highlight TNF as a candidate therapeutic target to control TET2-mutant CHIP.

Utilizing epigenetic regulators to improve HSC-based lentiviral gene therapy

AbstractThe curative benefits of autologous and allogeneic transplantation of hematopoietic stem cells (HSCs) have been proven in various diseases. However, the low number of true HSCs that can be collected from patients and the subsequent in vitro maintenance and expansion of true HSCs for genetic correction remains challenging. Addressing this issue, we here focused on optimizing culture conditions to improve ex vivo expansion of true HSCs for gene therapy purposes. In particular, we explored the use of epigenetic regulators to enhance the effectiveness of HSC-based lentiviral (LV) gene therapy. The histone deacetylase inhibitor quisinostat and bromodomain inhibitor CPI203 each promoted ex vivo expansion of functional HSCs, as validated by xenotransplantation assays and single-cell RNA sequencing analysis. We confirmed the stealth effect of LV transduction on the loss of HSC numbers in commonly used culture protocols, whereas the addition of quisinostat or CPI203 improved the expansion of HSCs in transduction protocols. Notably, we demonstrated that the addition of quisinostat improved the LV transduction efficiency of HSCs and early progenitors. Our suggested culture conditions highlight the potential therapeutic effects of epigenetic regulators in HSC biology and their clinical applications to advance HSC-based gene correction.

Hematopoietic Function Restoration by Transplanting Bone Marrow Niches In Vivo Engineered Using Carbonate Apatite Honeycomb Bioreactors

Hematopoietic stem cell (HSC) transplantation is used to treat blood and immunodeficient diseases. HSC expansion techniques must be developed to prevent complications and ensure reliable therapeutic efficacy. Hence, several studies have attempted in vitro expansion of HSCs using scaffolds but failed to mimic the diverse and complex nature of HSC environments. Herein, an artificial HSC microenvironment, bone marrow (BM) niches is created, through in vivo engineering using carbonate apatite honeycomb scaffolds and the potential of these scaffolds in restoring lost hematopoietic function and immunity is investigated. BM niches are generated in every honeycomb channel, wherein HSCs are gradually aggregated. Compared to the actual BM, the scaffolds exhibit a 9.9‐ and 78‐fold increase in the number of stored CD45− CD34+ side scatterlow cells that are mainly considered HSCs at 8 and 12 weeks, respectively. The transplantation of the honeycomb scaffold containing HSCs and BM niches into immunocompromised mice increases peripheral blood chimerism and restores hematopoietic function and the number of immunocytes (monocytes and lymphocytes) to normal levels. This study contributes to the development of efficient HSC transplantation techniques. Additionally, in vivo‐engineered integrated tissues using honeycomb scaffolds can be used to elucidate the interplay between the BM niches and resident cells.

Hematopoietic stem cell division is governed by distinct RUNX1 binding partners.

A defined number of hematopoietic stem cell (HSC) clones are born during development and expand to form the pool of adult stem cells. An intricate balance between self-renewal and differentiation of these HSCs supports hematopoiesis for life. HSC fate is determined by complex transcription factor networks that drive cell-type specific gene programs. The transcription factor RUNX1 is required for definitive hematopoiesis, and mutations in Runx1 have been shown to reduce clonal diversity. The RUNX1 cofactor, CBFý, stabilizes RUNX1 binding to DNA, and disruption of their interaction alters downstream gene expression. Chemical screening for modulators of Runx1 and HSC expansion in zebrafish led us to identify a new mechanism for the RUNX1 inhibitor, Ro5-3335. We found that Ro5-3335 increased HSC divisions in zebrafish, and animals transplanted with Ro5-3335 treated cells had enhanced chimerism compared to untreated cells. Using human CD34+ cells, we show that Ro5-3335 remodels the RUNX1 transcription complex by binding to ELF1, independent of CBFý. This allows specific expression of cell cycle and hematopoietic genes that enhance HSC self-renewal and prevent differentiation. Furthermore, we provide the first evidence to show that it is possible to pharmacologically increase the number of stem cell clones in vivo , revealing a previously unknown mechanism for enhancing clonal diversity. Our studies have revealed a mechanism by which binding partners of RUNX1 determine cell fate, with ELF transcription factors guiding cell division. This information could lead to treatments that enhance clonal diversity for blood diseases.

Genomic evolution of patients with myeloid neoplasms and known antecedent clonal hematopoiesis.

6584 Background: Clonal hematopoiesis (CH) refers to the clonal expansion of hematopoietic stem cells (HSCs) with one or more somatic mutations in the absence of a hematologic malignancy. Although it is associated with normal aging, CH confers an increased risk of several comorbidities and hematologic malignancies, including myeloid neoplasms (MNs). However, the outcomes of patients with CH that transformed to MN are unknown. Here, we aim to evaluate the clinicopathologic characteristics and survival outcomes of patients with MN with known antecedent CH. Methods: We retrospectively evaluated patients seen at a tertiary cancer center from May 2016 to July 2023 with myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), or acute myeloid leukemia (AML) and identified those who had CH prior to MN diagnosis. At the time of MN transformation, genomic data was extracted from whole bone marrow aspirate samples and subject to 81-gene target PCR-based sequencing using a next-generation sequencing platform. Results: A total of 36 patients were identified; at the time of CH diagnosis, 79% had known cardiovascular comorbidities and 81% had a prior diagnosis of non-myeloid malignancies for which 72% received prior chemotherapy and 25% radiation. According to the Clonal Hematopoiesis Risk Score (CHRS), 22% were low, 56% deemed intermediate, and 22% at high risk of transformation to MNs. The most frequently observed CH-associated mutations were TET2 (41%), TP53 (38%), IDH2 (16%), EZH2 (16%), ASXL1 (13%), and DNMT3A (13%); 69% had more than one somatic mutation, and 61% had normal cytogenetics. The median time to transformation to MN was 15 months, 26 months, and 24 months for MDS (n=28), CMML (n=2), and AML (n=6) patients, respectively (p=0.72). The median overall survival from MN diagnosis was 54 months, not reached, and 2 months for MDS, CMML, and AML, respectively (p=0.0015). We further delved into the genomic evolution from CH to MN. In the 6 pts who transformed to AML, 4 had a TP53 mutation at CH with expansion of the clone at MN transformation, 2 acquired NPM1 mutations and 1 acquired FLT3 mutations. Both pts who progressed to CMML had expansion of their underlying CH clones (KRAS and ZRSR2). In those who developed MDS, 22 pts had expansion of their CH-mutated clone at the time of MN, most frequently in TET2 (36%), IDH2 (18%), and TP53 (14%); 5 pts acquired new driver mutations outside of their CH clone; and 1 pt lost their CH clone with no new somatic mutations detected. Conclusions: Individuals with CH are at risk of progressing to MN, especially with external selective pressures like antineoplastic therapy that are not accounted for in prognostic systems like CHRS. Patients often have underlying high-risk clones at the time of CH diagnosis that expand at the time of MN, though some patients acquired new driver mutations.

Clonal hematopoiesis of indeterminate potential (CHIP): Outcomes and adverse events in patients with solid tumors.

3521 Background: CHIP is the clonal expansion of hematopoietic stem cells following acquisition of somatic mutations. It is a common incidental finding in cell-free DNA (cfDNA). We investigated the incidence of CHIP in cfDNA from patients with solid tumors and explored its association with outcomes and adverse events (AEs). Methods: We reviewed cfDNA results from a local prospective solid tumor cohort (PREDICT-L) and two randomized trials: CCTG CO.26 (durvalumab + tremelimumab [D+T] or best supportive care [BSC] in metastatic colorectal cancer [mCRC]) and CCTG PA.7 (gemcitabine and nab-paclitaxel [GN] +/- D+T in metastatic pancreatic adenocarcinoma [mPDAC]). CHIP+ was defined as any mutation in DNMT3A, TET2, ASXL1or ATM with a variant allele frequency (VAF) 2% or higher. Variants were considered germline if the VAF was &gt;40%, or were annotated as germline by respective assays. The first line of treatment after cfDNA was reviewed for grade 3 or higher and dose-limiting AEs. Results: The prevalence of CHIP was 10%-21% and was more common in older patients (p=0.003). There was no association between CHIP and sex (p=0.80) or ECOG status (p=0.31). The VAF of CHIP variants was similar across all panels, and DNMT3A was the gene most frequently mutated in all cohorts. Patients with CHIP in PA.7 treated with immunotherapy (IO) had improved progression-free survival (PFS) versus patients without CHIP (p=0.036, p-interaction=0.061 [multivariable]). However, patients with CHIP treated with chemotherapy (Chemo) showed a trend towards worse overall survival (OS) in the PREDICT-L cohort (p=0.057). There was no significant difference in the rates of AEs between the CHIP+ versus CHIP- groups for those treated with Chemo or IO. Conclusions: CHIP is common in patients with solid tumors. Although not appearing to impact rates of AEs, CHIP may impact outcomes from Chemo or IO. [Table: see text]

Clonal Hematopoiesis is Associated With Severe Cytokine Release Syndrome in Patients Treated With Chimeric Antigen Receptor T-Cell (CART) Therapy

Among patients receiving CD19 or B-cell maturation antigen (BCMA) CAR T therapy, inflammation pre- and post-CAR T infusion is implicated in the development of toxicities including cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and likely contributes to prolonged cytopenias. Clonal hematopoiesis (CH), the clonal expansion of hematopoietic stem cells harboring somatic mutations, has been associated with inflammasome upregulation. Herein, we examined the prevalence of pre-CAR T CH in a predominantly transplant-naïve cohort of recipients with non-Hodgkin lymphoma (NHL) or multiple myeloma (MM), and assessed the relationship between the presence of CH mutations and CAR T-related outcomes including CRS, ICANS, prolonged cytopenia, progression-free survival (PFS), and overall survival (OS). This study included 62 patients with NHL or MM who underwent CD19 or BCMA CAR T therapy from 2017 to 2022 at City of Hope and had available pre-CAR T cryopreserved peripheral blood mononuclear cells (PBMCs). DNA was isolated with QIAamp DNA Mini Kit (Qiagen) from PBMC samples (94% collected <30d of CART infusion), on which we performed targeted exome sequencing (108 pre-defined gene panel with 1000x sequencing depth) to determine the presence of CH (variant allele frequency [VAF] ≥2%). Multivariable logistic regression was used to examine the association between CH and absolute neutrophil count (ANC) recovery at day +30 and +60, maximum grade CRS and ICANS, grade <2 versus 2+, and OS and PFS at 1y. Covariates considered were age at CART, baseline ANC, sex, race, CAR-HEMATOTOX, LDH, bridging therapy (Y/N), and number of prior lines of therapy. Fifteen (24%) patients had at least one pathogenic CH mutation; 2 (13%) had ≥2 CH mutations concurrently. DMT3A mutations were the most common; 29% of mutations had VAFs >10%. Patients with CH were significantly more likely to develop grade ≥2 CRS (60% versus 28%, p = .03) compared to those without CH (odds ratio [OR] 3.9, 95% CI 1.2-13.2; p = .027). Accounting for baseline ANC (which was higher among the CH cohort and associated with delayed ANC recovery, p = .02) patients with CH did not have a significantly different rate of delayed ANC recovery compared to those without CH (adjusted OR 0.37, 95% CI 0.09-1.5; p = .17). There was no association between CH and ICANS, nor with 1y PFS or OS. CH was frequent (24%) in this cohort of CAR T recipients and was associated with a higher risk of development of grade ≥2 CRS after CAR T. Additional validation studies are currently underway, which may set the stage for consideration of pre-CAR T CH as a biomarker for risk stratification towards more proactive CRS prophylaxis. Translational studies could aim to prove a direct relationship between CH-mutated myeloid cells and CRS.

Chromatin modifier Hmga2 promotes adult hematopoietic stem cell function and blood regeneration in stress conditions

The molecular mechanisms governing the response of hematopoietic stem cells (HSCs) to stress insults remain poorly defined. Here, we investigated effects of conditional knock-out or overexpression of Hmga2 (High mobility group AT-hook 2), a transcriptional activator of stem cell genes in fetal HSCs. While Hmga2 overexpression did not affect adult hematopoiesis under homeostasis, it accelerated HSC expansion in response to injection with 5-fluorouracil (5-FU) or in vitro treatment with TNF-α. In contrast, HSC and megakaryocyte progenitor cell numbers were decreased in Hmga2 KO animals. Transcription of inflammatory genes was repressed in Hmga2-overexpressing mice injected with 5-FU, and Hmga2 bound to distinct regions and chromatin accessibility was decreased in HSCs upon stress. Mechanistically, we found that casein kinase 2 (CK2) phosphorylates the Hmga2 acidic domain, promoting its access and binding to chromatin, transcription of anti-inflammatory target genes, and the expansion of HSCs under stress conditions. Notably, the identified stress-regulated Hmga2 gene signature is activated in hematopoietic stem progenitor cells of human myelodysplastic syndrome patients. In sum, these results reveal a TNF-α/CK2/phospho-Hmga2 axis controlling adult stress hematopoiesis.