Normal Hematopoietic Stem Cells Research Articles

Characterization of the Human Mesenchymal Stromal Compartment in Normal Homeostasis and Acute Myeloid Leukemia at the Level of the Single Cell

Mesenchymal stromal cells (MSCs) constitute a pivotal component of the bone marrow niche, providing key regulatory signals required for maintenance of normal hematopoietic stem cell (HSC) function. In recent years, there has been an increased appreciation of the role played by the bone marrow niche in leukemogenesis with increasing evidence pointing towards dysregulation of MSCs in acute myeloid leukemia (AML). However, studies to date have predominantly evaluated culture expanded MSCs which do not necessarily reflect the state of MSCs in their in situ milieu. This is further compounded by limitations inherent to bulk population measurements which fall short of capturing the underlying heterogeneity that may exist among seemingly identical cells. Leveraging the combined powers of single cell RNA sequencing (scRNA-seq) and mass cytometry (CyTOF), we provide here an in-depth characterization of the human bone marrow MSC compartment both in normal homeostasis and AML. We address the question of MSC heterogeneity, and through integrated analysis of our scRNA-seq data and recently published publicly available datasets, we show that MSCs in their native state are transcriptionally split into subsets of varying differentiation potential that exist along a continuum of cellular states. These subsets consist of a predominant cluster showing high expression of genes implicated in adipogenesis and a smaller cluster demonstrating upregulation of osteogenesis-related genes, suggesting that these cells are poised toward adipogenic and osteogenic differentiation respectively. In addition, we observe a much smaller population with a gene expression profile in keeping with that of more terminally differentiated osteoblasts, however this is not consistently observed across all samples. Furthermore, our comparative analysis of publicly available scRNA-seq datasets of murine stroma shows a high degree of conservation with similar patterns of cellular diversity. This pattern of cellular architecture is however lost in culture-expanded MSCs, underscoring the limitations associated with their use. In the context of AML, we show that native MSCs undergo transcriptional transformation, acquiring a distinct gene signature pattern distinguishing them from their normal counterparts. Among these changes are upregulation of genes implicated in inflammation indicating that deregulation of inflammatory signalling in AML is facilitated, at least in part, by cell-extrinsic mechanisms. In summary, our data reveal novels insights into the cellular architecture of the human MSC compartment in its native, unmanipulated state as well as unravel key transcriptional changes that characterize MSCs in AML, which can potentially be exploited for therapeutic targeting.

Highly efficient Runx1 enhancer eR1-mediated genetic engineering for fetal, child and adult hematopoietic stem cells

A cis-regulatory genetic element which targets gene expression to stem cells, termed stem cell enhancer, serves as a molecular handle for stem cell-specific genetic engineering. Here we show the generation and characterization of a tamoxifen-inducible CreERT2 transgenic (Tg) mouse employing previously identified hematopoietic stem cell (HSC) enhancer for Runx1, eR1 (+24 m). Kinetic analysis of labeled cells after tamoxifen injection and transplantation assays revealed that eR1-driven CreERT2 activity marks dormant adult HSCs which slowly but steadily contribute to unperturbed hematopoiesis. Fetal and child HSCs that are uniformly or intermediately active were also efficiently targeted. Notably, a gene ablation at distinct developmental stages, enabled by this system, resulted in different phenotypes. Similarly, an oncogenic Kras induction at distinct ages caused different spectrums of malignant diseases. These results demonstrate that the eR1-CreERT2 Tg mouse serves as a powerful resource for the analyses of both normal and malignant HSCs at all developmental stages.

Hematopoietic and Chronic Myeloid Leukemia Stem Cells: Multi-Stability versus Lineage Restriction.

There is compelling evidence to support the view that the cell-of-origin for chronic myeloid leukemia is a hematopoietic stem cell. Unlike normal hematopoietic stem cells, the progeny of the leukemia stem cells are predominantly neutrophils during the disease chronic phase and there is a mild anemia. The hallmark oncogene for chronic myeloid leukemia is the BCR-ABLp210 fusion gene. Various studies have excluded a role for BCR-ABLp210 expression in maintaining the population of leukemia stem cells. Studies of BCR-ABLp210 expression in embryonal stem cells that were differentiated into hematopoietic stem cells and of the expression in transgenic mice have revealed that BCR-ABLp210 is able to veer hematopoietic stem and progenitor cells towards a myeloid fate. For the transgenic mice, global changes to the epigenetic landscape were observed. In chronic myeloid leukemia, the ability of the leukemia stem cells to choose from the many fates that are available to normal hematopoietic stem cells appears to be deregulated by BCR-ABLp210 and changes to the epigenome are also important. Even so, we still do not have a precise picture as to why neutrophils are abundantly produced in chronic myeloid leukemia.

Cell-intrinsic factors governing quiescence vis-à-vis activation of adult hematopoietic stem cells.

Hematopoiesis is a highly complex process, regulated by both intrinsic and extrinsic factors. Often, these two regulatory arms work in tandem to maintain the steady-state condition of hematopoiesis. However, at times, certain intrinsic attributes of hematopoietic stem cells (HSCs) override the external stimuli and dominate the outcome. These could be genetic events like mutations or environmentally induced epigenetic or transcriptomic changes. Since leukemic stem cells (LSCs) share molecular pathways that also regulate normal HSCs, identifying specific, dominantly acting intrinsic factors could help in the development of novel therapeutic approaches. Here we have reviewed such dominantly acting intrinsic factors governing quiescence vis-à-vis activation of the HSCs in the face of external forces acting on them. For brevity, we have restricted our review to the articles dealing with adult HSCs of human and mouse origin that have been published in the last 10years. Hematopoietic stem cells (HSCs) are closely associated with various stromal cells in their microenvironment and, thus, constantly receive signaling cues from them. The illustration depicts some dominantly acting intrinsic or cell-autonomous factors operative in the HSCs. These fall into various categories, such as epigenetic regulators, transcription factors, cell cycle regulators, tumor suppressor genes, signaling pathways, and metabolic regulators, which counteract the outcome of extrinsic signaling exerted by the HSC niche.

PRL2 phosphatase enhances oncogenic FLT3 signaling via dephosphorylation of the E3 ubiquitin ligase CBL at tyrosine 371

AbstractAcute myeloid leukemia (AML) is an aggressive blood cancer with poor prognosis. FMS-like tyrosine kinase receptor-3 (FLT3) is one of the major oncogenic receptor tyrosine kinases aberrantly activated in AML. Although protein tyrosine phosphatase PRL2 is highly expressed in some subtypes of AML compared with normal human hematopoietic stem and progenitor cells, the mechanisms by which PRL2 promotes leukemogenesis are largely unknown. We discovered that genetic and pharmacological inhibition of PRL2 significantly reduce the burden of FLT3-internal tandem duplications–driven leukemia and extend the survival of leukemic mice. Furthermore, we found that PRL2 enhances oncogenic FLT3 signaling in leukemia cells, promoting their proliferation and survival. Mechanistically, PRL2 dephosphorylates the E3 ubiquitin ligase CBL at tyrosine 371 and attenuates CBL-mediated ubiquitination and degradation of FLT3, leading to enhanced FLT3 signaling in leukemia cells. Thus, our study reveals that PRL2 enhances oncogenic FLT3 signaling in leukemia cells through dephosphorylation of CBL and will likely establish PRL2 as a novel druggable target for AML.

Lipids and the cancer stemness regulatory system in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease of impaired myeloid differentiation and a caricature of normal hematopoiesis. Leukemic stem cells (LSCs) are responsible for long-term clonal propagation in AML just as hematopoietic stem cells (HSCs) sustain lifelong hematopoiesis. LSCs are often resistant to standard chemotherapy and are responsible for clinical relapse. Although AML is highly heterogeneous, determinants of stemness are prognostic for AML patient survival and can predict AML drug sensitivity. Therefore, one way to overcome challenges preventing efficacious treatment outcomes is to target LSC stemness. Metabolomic and lipidomic studies of serum and cells from AML patients are emerging to complement genomic, transcriptomic, epigenetic, and proteomic data sets to characterize and stratify AML. Recent studies have shown the value of fractionating LSCs versus blasts when characterizing metabolic pathways and implicate the importance of lipid balance to LSCs function. As more extensive metabolic studies coupled to functional in vivo assays are conducted on highly purified HSCs, bulk AML, and LSCs, the similarities and differences in lipid homeostasis in stem-like versus more mature AML subtypes as well as from normal HSCs are emerging. Here, we discuss the latest findings from studies of lipid function in LSCs, with a focus on sphingolipids (SLs) as stemness/lineage fate mediators in AML, and the balance of fatty acid anabolism and catabolism fueling metabolic flexibility and drug resistance in AML. We also discuss how designing successful strategies to target lipid vulnerabilities and improve AML patient survival should take into consideration the hierarchical nature of AML.

The development of therapy related myeloid neoplasms in childhood cancer survivors

Childhood cancer survivors exposed to chemotherapy show signs of accelerated aging and are at risk of developing secondary malignancies; however, the mechanisms responsible for these long-term adverse effects are not clear. In a recent study, Bertrums et al. show that exposure to chemotherapy results in an increase in mutational age of normal hematopoietic stem cells.

622MO An asymmetrical CLL1/CD3 bispecific antibody, ABL602, exhibits CLL1 binding-dependent CD3 binding/activation and antitumor activity in acute myeloid leukemia (AML) mouse model and leukemia blasts from AML patients

C-type lectin-like molecule 1 (CLL1) is highly expressed in acute myeloid leukemia blast cells but not in normal hematopoietic stem cells (HSCs). This limited expression of CLL1 makes it as a promising target for the AML therapy compared with other targets such as CD33 and CD123, which are also expressed in HSCs. Previously we have shown potent CLL1-specific antitumor activity using two AML cell line/models. In this study, we determined whether binding affinity of ABL602 to CD3 can be augmented upon binding to CLL1 and expanded our study to various AML cell lines and blasts from AML patients. To determine CLL1 binding-dependent CD3 binding of ABL602, T cell binding was measured by flow cytometry in the presence of CLL1-spiked beads. ABL602-mediated antitumor activity and T cell activation were tested in CLL1 expressing AML cell lines and AML blasts from patients. In vivo antitumor activity was determined in humanized mice bearing AML tumors. ABL602 (2+1), with its CD3 binding arm sterically hindered by CLL1 binding arm, binds to CD3 with a lower affinity compared with 1+1 format in the absence of CLL1-expressing cells. By incubating T cells with beads spiked with various amounts of CLL1 in the presence of ABL602, we proved that ABL602’s binding affinity to CD3 was increased in a CLL1-dependent manner, reaching comparable level to that of 1+1. ABL602 induced superior tumor cell killing than 1+1 reference antibody JNJ-67571244. Moreover, ABL602 showed a dose-dependent anti-tumor activity with tumor regression in OCIAML2 xenograft and HL-60 orthotopic model. In AML patient blasts, CLL1 was highly expressed and ABL602 exhibited a strong T cell activation and tumor killing activity on CLL1+ AML blasts. This study supports the target-specific T cell activation and tumor killing activity of 2+1 heterodimeric structure of ABL602 on both AML cell lines and patient-originated AML blasts and warrants further pre-clinical development.

Degradation of GSPT1 causes TP53-independent cell death in leukemia while sparing normal hematopoietic stem cells.

Targeted protein degradation is a rapidly advancing and expanding therapeutic approach. Drugs that degrade GSPT1 via the CRL4CRBN ubiquitin ligase are a new class of cancer therapy in active clinical development with evidence of activity against acute myeloid leukemia in early-phase trials. However, other than activation of the integrated stress response, the downstream effects of GSPT1 degradation leading to cell death are largely undefined, and no murine models are available to study these agents. We identified the domains of GSPT1 essential for cell survival and show that GSPT1 degradation leads to impaired translation termination, activation of the integrated stress response pathway, and TP53-independent cell death. CRISPR/Cas9 screens implicated decreased translation initiation as protective following GSPT1 degradation, suggesting that cells with higher levels of translation are more susceptible to the effects of GSPT1 degradation. We defined 2 Crbn amino acids that prevent Gspt1 degradation in mice, generated a knockin mouse with alteration of these residues, and demonstrated the efficacy of GSPT1-degrading drugs in vivo with relative sparing of numbers and function of long-term hematopoietic stem cells. Our results provide a mechanistic basis for the use of GSPT1 degraders for the treatment of cancer, including TP53-mutant acute myeloid leukemia.

Inhibitor of DNA binding proteins revealed as orchestrators of steady state, stress and malignant hematopoiesis.

Adult mammalian hematopoiesis is a dynamic cellular process that provides a continuous supply of myeloid, lymphoid, erythroid/megakaryocyte cells for host survival. This process is sustained by regulating hematopoietic stem cells (HSCs) quiescence, proliferation and activation under homeostasis and stress, and regulating the proliferation and differentiation of downstream multipotent progenitor (MPP) and more committed progenitor cells. Inhibitor of DNA binding (ID) proteins are small helix-loop-helix (HLH) proteins that lack a basic (b) DNA binding domain present in other family members, and function as dominant-negative regulators of other bHLH proteins (E proteins) by inhibiting their transcriptional activity. ID proteins are required for normal T cell, B cell, NK and innate lymphoid cells, dendritic cell, and myeloid cell differentiation and development. However, recent evidence suggests that ID proteins are important regulators of normal and leukemic hematopoietic stem and progenitor cells (HSPCs). This chapter will review our current understanding of the function of ID proteins in HSPC development and highlight future areas of scientific investigation.

Chimeric Antigen Receptor T Cells Targeting Cell Surface GRP78 to Eradicate Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a serious, life-threatening hematological malignancy. The treatment outcome of relapsed or refractory AML patients remains dismal, and new treatment options are needed. Chimeric antigen receptor (CAR) T cells have been successful in improving the prognosis for B-lineage acute lymphoblastic leukemia and lymphoma by targeting CD19. However, CAR T-cell therapy for AML is still elusive, owing to the lack of a tumor-specific cell surface antigen and spare hematopoietic stem cells (HSCs). This study generated a novel CAR construction that targets the cell surface protein glucose-regulated protein 78 (GRP78) (csGRP78). We confirmed that GRP78-CAR T cells demonstrate an anti-tumor effect against human AML cells in vitro. In xenograft models, GRP78-CAR T cells effectively eliminate AML cells and protect mice against systemic leukemia, in the meanwhile, prolonging survival. In addition, GRP78-CAR T cells also specifically eradicate the primary AML patient-derived blast. In particular, GRP78-CAR T cells spare normal HSCs, highlighting that GRP78-CAR is a promising approach for the therapy of AML.

Proteomic Profiling Identifies Specific Leukemic Stem Cell-Associated Protein Expression Patterns in Pediatric AML Patients.

Simple SummaryAcute myeloid leukemia is an aggressive cancer in children and novel therapeutic tools are warranted to improve outcomes and reduce late effects in these patients. In this study, we isolate and explore the protein profiles of leukemic stem cells and normal hematopoietic stem cells from hematologically healthy children. Differences in protein profiles between leukemic and normal hematopoietic stem cells were identified. These results provide an insight into the disrupted biological pathways in childhood acute myeloid leukemia. Moreover, differences in protein profiles may serve as potential targets for future therapies specifically aiming at the disease-propagating leukemic stem cells while omitting the normal hematopoietic stem cells.Novel therapeutic tools are warranted to improve outcomes for children with acute myeloid leukemia (AML). Differences in the proteome of leukemic blasts and stem cells (AML-SCs) in AML compared with normal hematopoietic stem cells (HSCs) may facilitate the identification of potential targets for future treatment strategies. In this explorative study, we used mass spectrometry to compare the proteome of AML-SCs and CLEC12A+ blasts from five pediatric AML patients with HSCs and hematopoietic progenitor cells from hematologically healthy, age-matched controls. A total of 456 shared proteins were identified in both leukemic and control samples. Varying protein expression profiles were observed in AML-SCs and leukemic blasts, none having any overall resemblance to healthy counterpart cell populations. Thirty-four proteins were differentially expressed between AML-SCs and HSCs, including the upregulation of HSPE1, SRSF1, and NUP210, and the enrichment of proteins suggestive of protein synthesis perturbations through the downregulation of EIF2 signaling was found. Among others, NUP210 and calreticulin were upregulated in CLEC12A+ blasts compared with HSCs. In conclusion, the observed differences in protein expression between pediatric patients with AML and pediatric controls, in particular when comparing stem cell subsets, encourages the extended exploration of leukemia and AML-SC-specific biomarkers of potential relevance in the development of future therapeutic options in pediatric AML.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Targeting stem cells in myelodysplastic syndromes and acute myeloid leukemia.

The genetic architecture of cancer has been delineated through advances in high‐throughput next‐generation sequencing, where the sequential acquisition of recurrent driver mutations initially targeted towards normal cells ultimately leads to malignant transformation. Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are hematologic malignancies frequently initiated by mutations in the normal hematopoietic stem cell compartment leading to the establishment of leukemic stem cells. Although the genetic characterization of MDS and AML has led to identification of new therapeutic targets and development of new promising therapeutic strategies, disease progression, relapse, and treatment‐related mortality remain a major challenge in MDS and AML. The selective persistence of rare leukemic stem cells following therapy‐induced remission implies unique resistance mechanisms of leukemic stem cells towards conventional therapeutic strategies and that leukemic stem cells represent the cellular origin of relapse. Therefore, targeted surveillance of leukemic stem cells following therapy should, in the future, allow better prediction of relapse and disease progression, but is currently challenged by our restricted ability to distinguish leukemic stem cells from other leukemic cells and residual normal cells. To advance current and new clinical strategies for the treatment of MDS and AML, there is a need to improve our understanding and characterization of MDS and AML stem cells at the cellular, molecular, and genetic levels. Such work has already led to the identification of promising new candidate leukemic stem cell molecular targets that can now be exploited in preclinical and clinical therapeutic strategies, towards more efficient and specific elimination of leukemic stem cells.

Chimeric antigen receptor T-cells targeting IL-1RAP: a promising new cellular immunotherapy to treat acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) remains a very difficult disease to cure due to the persistence of leukemic stem cells (LSCs), which are resistant to different lines of chemotherapy and are...

COVID-19 vaccination antibody responses in patients with aplastic anaemia and paroxysmal nocturnal haemoglobinuria

COVID-19 vaccination antibody responses in patients with aplastic anaemia and paroxysmal nocturnal haemoglobinuria

P438: ARYL HYDROCARBON RECEPTOR SIGNALLING IN NORMAL HAEMATOPOIETIC (HSC) AND LEUKEMIA STEM CELLS (LSC): DIFFERENTIAL EFFECT OF AHR ANTAGONIST ON LSC COMPARED TO HSC

Background: Acute myeloid leukaemia (AML) is a devastating disease with poor prognosis. It is driven by leukemia stem cells (LSCs), which are able to survive treatment and drive relapse. This highlights the importance of characterizing LSCs. LSC-targeted treatment strategies are limited because there are no known surface markers that are specific for LSCs in every leukaemia. Targeting a signalling pathway that is altered in all LSCs could be a novel, promising treatment strategy. The aryl hydrocarbon receptor (AHR) pathway was shown to be critical for the self-renewal properties of normal haematopoietic stem cells (HSCs). Aims: We aimed to study the consequences of manipulating the AHR pathway in vivo in a murine leukaemia model. Methods: We used the following methods: murine bone marrow transplantation leukemia model (MBMTLM) - CALM-AF10 with limiting dilution assay (LDA); competitive repopulation assay (CRA) with LDA (CD45.2 donors into CD45.1 recipient mice), transcriptome analysis (sequencing with the Illumina platform and analisys with the DESeq2). Treatment of the cells was performed at various experiments with the AHR agonist, ITE, and an AHR antagonist, CH-223191 (a vehicle control is DMSO). Results: We used a murine retroviral transduction bone marrow transplantation leukemia model (MBMTLM)) with the CALM-AF10 minimal fusion gene (CAMF) as the oncogenic driver, to study the effect of AHR agonist (ITE) and an AHR antagonist (CH-223191) treatment on LSCs. Interestingly, both AHR agonist and antagonist treatment affected LSC frequency and survival of mice transplanted with treated cells. The LSC frequency as measured by limiting dilution transplantation assays (LDA) was 3.7 times lower after agonist treatment (1:163, blue survival curve in fig. 1, A) compared to a vehicle control (1:44, DMSO, black survival curve)). Very interestingly, after antagonist treatment the LSC frequency was so low that could not be measured as none of the mice transplanted with antagonist-treated cells in the LDAs developed leukaemia (n=9, CH-223191 (red curve in fig. 1, A)). Of note, our CAMF MBMTLM has a very high LSC frequency of about 1:4 in cells that were not cultured after thawing. We performed RNA-Seq on 90 samples to determine changes in gene expression patterns after treatment of healthy bone marrow cells and CAMF leukemia cells with the AHR agonist and antagonist. Preliminary differential expression analysis with DESeq2 revealed differential expression of genes involved in apoptosis and proliferation in leukaemia cells treated with CH-223191. As leukemia did not develop in recipients of cells treated with the antagonist CH-223191, genes differentially regulated in these cells might be critical and specific to LSC function and will be candidate targets for LSC-specific treatments. To confirm that AHR antagonist treatment affects LSCs differently than normal HCS, we performed competitive repopulation assay (CRA). Our preliminary data (n = 12) show that treatment of HSCs with CH-223191 results in a steady increase in the engraftment over a 47-week period following transplantation (coral bars, fig. 1, B), as opposed to the grafts from the ITE-treated arm (blue bars), vehicle or untreated HSCs (black and green bars, respectively). Image:Summary/Conclusion: In summary, our data show that the LSCs react differently than normal HSCs to changes in AHR signalling. These differences suggest it might be possible to specifically inhibit LSCs while sparing normal HSCs, thus paving the way to develop strategies to treat leukemia based on manipulating AHR signalling.

P582: FIRST RESULTS OF A PHASE II STUDY (STIMULUS-AML1) INVESTIGATING SABATOLIMAB + AZACITIDINE + VENETOCLAX IN PATIENTS WITH NEWLY DIAGNOSED ACUTE MYELOID LEUKEMIA

Background: The BCL-2 inhibitor venetoclax (VEN) in combination with a hypomethylating agent (HMA) has improved outcomes for patients with newly diagnosed (ND) acute myeloid leukemia (AML) who are unfit for intensive chemotherapy (IC; DiNardo CD, N Engl J Med, 2020); however, responses are often transient. Sabatolimab is a novel immuno-myeloid therapy targeting TIM-3, an immune regulator expressed on immune cells and myeloid leukemic progenitors but not on normal hematopoietic stem cells. Sabatolimab + HMA has shown promising durable responses in a Phase Ib study in patients with ND-AML and myelodysplastic syndrome (Wei AH, ASH 2021; NCT03066648). Treatment with sabatolimab + VEN + azacitidine (AZA) may also induce different pathways of cancer cell elimination. Aims: In this first presentation of the study, we report findings from the dose-escalation part of STIMULUS-AML1 (NCT04150029), a Phase II, single-arm study of sabatolimab + AZA + VEN in adult patients with AML. Methods: Adult patients with ND-AML ineligible for IC were enrolled. In dose-escalation part 1 (safety run-in), patients received either 400 mg (Cohort 1) or 800 mg (Cohort 2) sabatolimab every 4 weeks (Q4W) plus 400 mg VEN once a day every day, plus 75 mg/m2 AZA on Days (D) 1-7, or D1-5+D8-9, or D1-6+D8 of a 28-day cycle (Zeidan A, ESH-AML 2022). For the safety run-in part, the primary endpoint was assessment of the incidence of dose-limiting toxicities (DLTs) between Cycle 1 D8 and the end of Cycle 2. Results: As of the Sep 6, 2021 data cutoff, 18 patients have been treated in part 1 of the study with sabatolimab + AZA + VEN (Cohort 1, n=5; Cohort 2, n=13). Baseline demographics are shown in Table 1. Treatment was ongoing for 9 (50%) patients (Cohort 1, n=1; Cohort 2, n=8). Reasons for study treatment discontinuation included adverse events (AEs; n=3), progressive disease (n=3, including a case of chloroma), planned stem cell transplant (n=2), and physician decision (n=1). Only 1 DLT (elevated troponin T/asymptomatic myocarditis) was reported in Cohort 2 and no DLTs were reported in Cohort 1. Table 2 summarizes AEs observed in >20% of patients in the study. Consistent with AEs often observed during AZA + VEN therapy (DiNardo CD, N Engl J Med, 2020), the 5 most common AEs regardless of relationship to study treatment were constipation (all grades [gr]: 39%; gr ≥3: 0%) and hematologic events, including anemia (33%; 28%), decreased platelets (33%; 33%), neutropenia (39%; 39%), and febrile neutropenia (gr: 50%). Treatment-related AEs gr ≥3 in >25% of all patients included neutropenia (39%), decreased platelet count (33%), and decreased neutrophil count (28%). Treatment-related AEs led to study treatment discontinuation in 3 patients (platelet count decreased [n=2], troponin level increased/asymptomatic myocarditis [n=1]). AEs led to sabatolimab dose interruption in 6 patients, all in Cohort 2. No sabatolimab dose reduction was observed. Fourteen patients had dose interruption of VEN due to AEs. Five patients had dose reduction of VEN (none due to AEs). Serious AEs were reported in 14 (78%) patients; the most common was febrile neutropenia (44%). No other serious AEs were reported in >1 patient. Efficacy data will be presented at the EHA meeting. Image:Summary/Conclusion: Safety and tolerability of sabatolimab + AZA + VEN were comparable at 2 dose levels (400 and 800 mg) of sabatolimab and were overall comparable to the reported safety profile of VEN + AZA doublet therapy. These findings supported initiation of the expansion cohort of STIMULUS-AML1 at a sabatolimab 800 mg dose.

P693: DIAGNOSTIC ROLE OF CD26+ LEUKEMIC STEM CELLS IN CHRONIC MYELOID LEUKEMIA

Background: Diagnosis of CML consists of persistent leucocytosis with the demonstration of the Philadelphia (Ph) chromosome abnormality, the t(9;22)(q34;q11), by routine cytogenetics, fluorescence in situ hybridization (FISH) or by molecular studies such as RT-PCR.These conventional methods suffer from a longer turn around time, higher costs and requirement of labs with adequate expertise. CD26 is a highly specific marker expressed in CML stem cells, which is not present on normal hematopoietic stem cells or on Leukemia stem cells (LSCs) of other myeloid neoplasms. A flow-cytometry based assessment of CD26+LSCs may prove to be an optimal biomarker for the diagnosis and monitoring of CML patients. Aims: 1)Assessment of CD 26 expression in suspected cases of CML-chronic phase 2)Correlation of CD 26+ stem cells with clinico-pathological parameters at baseline and its kinetics on tyrosine kinase inhibitor (TKI) treatment on further follow-up at 12 months. Methods: Suspected patients of CML-CP were included in the study. Peripheral blood were utilized for flowcytometric assessment of CD26+ LSCs. The results were correlated with conventional diagnostic tests of CML. Patients with myeloid neoplasms other than CML who proved negative for BCR-ABL1 by RT-PCR and normal HSCs donors treated with granulocyte-colony stimulating factors (G-CSF), were included as negative controls. All samples were processed using standard stain -lyse-wash method within 24 hr of collection. To reach a sensitivity of 10−5, acquisition of at least 1.0 × 106 cells was performed on FACS Canto II flow cytometer data a FACS DIVA 8.0.3 software (BD, Biosciences).CD26 expression was evaluated by a sequential gating strategy. After exclusion of doublets and debris CD45 dim to intermediate/CD34+/CD38- population was gated sequentially and then the expression of CD 26+ was studied on this population. All the samples were simultaneously assessed for BCR-ABL1 transcript by standard RT-PCR analysis. Patients on treatment were also followed up at 12 months to detect levels of CD26+ LSCs and correlated with BCR -ABL1 levels by qPCR. Results: A total of 86 patients of suspected cases of CML were included in the study from June 2020 to Dec 2021.The On flowcytometry analysis, CD 26 expression was present in 75 cases. All the cases with positive CD26+ LSCs were found to be positive on RT-PCR for BCR-ABL transcript. Eleven cases which were scored negative for CD26+ LSCs were also found to be negative for any BCR-ABL transcript. These negative cases included 8 cases of primary myelofibrosis, 2 cases of chronic myelomonocytic leukemia and 1 case of Polycythaemia Vera. The median percentage of CD26+ LSCs was 39.8% (Range-0.5-100). There was no significant correlation of the CD26 levels with age (p value=0.96), sex (p value=0.7), percentage of blasts (p value = 0.88), total leucocyte counts (p value=0.32), basophils (p value = 0.19), Sokal score (p value = 0.99) and ELTS score (p value = 0.99). Follow-up data was available for 7 patients and at 12 months all of these patients had achieved major molecular remission on TKIs. Two patients scored negative for CD26+LSCs while five patients showed significant fall in the level of CD 26+ LSCs however it was still detectable by flowcytometry (p value= 0.04). Summary/Conclusion: Flowcytometry assessment of CD26 + LSCs is a rapid and cheap diagnostic tool for diagnosis of CML with high diagnostic efficacy. Falling levels on TKIs warrants further research to prove it as a surrogate for monitoring response by conventional methods.

P787: SABATOLIMAB (MBG453) COMBINATION TREATMENT REGIMENS FOR PATIENTS WITH HIGHER-RISK MYELODYSPLASTIC SYNDROMES: THE MYELODYSPLASTIC SYNDROMES STUDIES IN THE STIMULUS IMMUNO-MYELOID CLINICAL TRIAL PROGRAM

Background: Patients (pts) with higher-risk myelodysplastic syndromes (HR-MDS) unfit for hematopoietic stem cell transplant (HSCT) have poor outcomes with single-agent hypomethylating agent (HMA) therapy. Novel treatments with durable responses and survival benefit are needed. TIM-3 regulates innate and adaptive immunity and is expressed on leukemic stem cells (LSCs) and blasts but not normal hematopoietic stem cells. Sabatolimab is a novel immuno-myeloid therapy that binds to TIM-3 on immune cells, facilitating immune activation and phagocytosis of leukemic cells. Sabatolimab also binds to TIM-3 on leukemic cells, potentially impeding self-renewal of LSCs. Sabatolimab + HMAs had promising efficacy and response durability in HR-MDS in early-phase (ph) trials, with few significant immunologic adverse events (AEs; Brunner AM. ASH 2021. Oral 244). Aims: The STIMULUS immuno-myeloid clinical trial program investigates the safety, efficacy, and durability of sabatolimab-based combination therapies in patients with myeloid malignancies. Exploration of sabatolimab’s mechanism of action and identification of potential biomarkers predictive of response are planned. This abstract summarizes the design of 4 ongoing STIMULUS trials in previously untreated adult pts with HR-MDS ineligible for intensive chemotherapy or HSCT. Methods: STIMULUS-MDS1 (NCT03946670) is an international, randomized, double-blind, placebo-controlled, ph II trial evaluating sabatolimab + HMA in pts with very-high, high-, or intermediate-risk (vH/H/IR)-MDS who have completed enrollment (N=127). Primary endpoints are complete remission (CR) and progression-free survival (PFS). STIMULUS-MDS2 (NCT04266301) is an international, randomized, double-blind, placebo-controlled, ph III trial of sabatolimab + azacitidine (AZA) in pts with vH/H/IR-MDS or chronic myelomonocytic leukemia-2; the study has completed recruiting (N=530). Primary endpoint is overall survival (OS). Key secondary endpoints are time to definitive deterioration of fatigue; red blood cell transfusion-free interval; and improvement of fatigue, physical, and emotional functioning. STIMULUS MDS-US (NCT04878432) is a US-based, open-label, single-arm, ph II trial of sabatolimab + HMA of investigator’s choice (AZA intravenous [IV] or subcutaneous, decitabine IV, or oral decitabine/cedazuridine) in pts with vH/H/IR-MDS. Target enrollment is 90 pts. Primary endpoint is safety. Secondary endpoints include CR, PFS, leukemia-free survival (LFS), and OS. STIMULUS-MDS3 (NCT04812548) is an international, open-label, single-arm, ph II trial that explores triplet therapy of sabatolimab + AZA + BCL-2 inhibitor venetoclax (VEN) in pts with vH/HR-MDS. Part 1 is a safety run-in comprising 2 safety cohorts with ~6 pts receiving sabatolimab 400 mg + AZA + VEN and ~12 pts receiving sabatolimab 800 mg + AZA + VEN. Primary endpoint of part 1 is safety. If (both 400- and 800-mg doses) sabatolimab + AZA + VEN are safe, the trial will move to an expansion cohort (~58 pts) of sabatolimab 800 mg Q4W + AZA + VEN. Primary endpoint is CR. Secondary endpoints include CR + mCR rate, overall response rate (CR + mCR + PR + hematologic improvement), OS, PFS, LFS, and event-free survival. The STIMULUS immuno-myeloid clinical trial program is investigating the efficacy and safety profiles and the quality-of-life improvements for sabatolimab-based combination therapies in pts with myeloid malignancies. The STIMULUS-MDS trials aim to gain insight into the durable benefit of sabatolimab combination therapies in pts with HR-MDS. Results: None. Image:Summary/Conclusion: None.