Normal Bone Marrow Research Articles

ИЗМЕРЯЕМЫЙ КОЭФФИЦИЕНТ ДИФФУЗИИ В ДИАГНОСТИКЕ МЕТАСТАТИЧЕСКОГО ПОРАЖЕНИЯ КОСТЕЙ СКЕЛЕТА

Aim: To evaluate the possibilities of absolute and relative values of the apparent diffusion coefficient (ADC) in the diagnosis of metastatic lesions of skeletal bones. Material and methods: The study included 12 patients with metastatic bone lesions, before any treatment was applied. The age of the patients ranged from 38 to 73 years, 3 men, 9 women. Among the morphological forms of tumors were presented: cancers of the breast (3), prostate (1) glands, colon (1), lung (2), body (1) and cervix (1) uterus, thyroid (1) and pancreas (2). The detected changes were classified on the basis of Bone scan, SPECT/CT and standard MRI. The ADC values of unchanged bone marrow (n=360), divided by anatomical zones, metastatic foci (n=117), as well as benign changes of various nature (n=19) were analyzed. The ratio of the ADC values of each of the metastatic and benign focal formations to the normal values for each of the localizations was calculated. Results: Unchanged bone marrow, depending on anatomical localization (cervical, thoracic, lumbar spine, pelvic bones, shoulder blades, collarbones, sternum, ribs, proximal humerus and femur bones) from the point of view of ADC, showed statistically significant heterogeneity. Statistical analysis has shown that there is no connection between the groups of ADC indicators in the foci of metastatic lesions, combined depending on the anatomical localization. With a similar comparison, but by belonging to the primary tumor, an even greater intergroup difference was found. ADC values in the foci of metastatic lesions turned out to be dependent on the morphological type of the primary tumor and significantly differ both from metastases of other morphological affiliation and from normal red bone marrow parameters. The use of relative values allowed to increase the specificity from 15 to 19 %. Conclusion: This study showed that knowledge of the range of reference ADC values for unchanged red bone marrow and anomalies of various genesis is important in differential diagnosis. The differentiation of the skeleton into separate anatomical zones probably makes it possible to increase the effectiveness of the isolated application of the technique when it comes to absolute values. The morphological affiliation of metastatic foci is important in the formation of ADC values, rather than the bone marrow microenvironment, which is also supported by the low efficiency of the use of relative values.

Stem-Cell Enriched Cellular Hierarchy of TP53 Mutant Acute Myeloid Leukemia Is Vulnerable to Targeted Protein Degradation of c-MYC

Deregulation of MYC genes occurs in up to 70% of all human cancers and is associated with hallmarks of cancer including mitochondrial and ribosomal biogenesis, cell cycle progression, and metabolic abnormalities. TP53 regulates MYC while MYC suppresses TP53, suggesting counteracting negative feedback loops. Therefore, MYC or its function can be activated when TP53 is not functional. TP53 mutations occur in 30% of relapsed/refractory acute myeloid leukemias (AMLs) patients' survival is dismal, and there are no effective therapies for these patients. Compared to TP53 wild-type (TP53wt), TP53 mutant (TP53mut) AMLs have lower percentages and numbers of leukemia blasts with increased immature CD34+ cells and resistance to chemo- or molecularly targeted therapies. However, the exact cellular hierarchy of TP53mut AML has not been elucidated. We observed significantly increased MYC mRNA levels in (TP53mut), as compared to TP53wt AML, and also increased levels in TP53mut versus TP53wt AML leukemia stem cell (LSC) fractions. This finding was confirmed in a dataset from the Munich Leukemia Laboratory (N = 732). We found significantly upregulated MYC pathways in TP53mut compared to TP53wt AML LSC. We confirmed the increased MYC mRNA levels at the protein level in TP53mut AML by single-cell mass cytometry (CyTOF). To dissect the cellular hierarchy in TP53mut AML, we performed single-cell RNA sequencing of 32 BM samples from healthy donors (N = 3), newly diagnosed, high-risk TP53wt (N = 7) and TP53mut (N = 22) AML patients, with 6,685, 10,687 and 10,687 cells from normal, TP53wt and TP53mut AML bone marrow (BM) cells, respectively. We found highly enriched HSC-like cells and reduced progenitor- and GMP-like cells in TP53mut AML compared to normal BM (NBM) and TP53wt AML samples. We overlayed MYC expression levels on the mapping of cellular components and found higher MYC levels in HSC-like cells in TP53mut AML compared to HSC and HSC-like cells in NBM and TP53wt AML samples, suggesting enrichment of immature HSC-like cells and increased activity of MYC in TP53mut AML LSCs ( Fig. A). To target c-MYC and MYC signaling, we utilized GT19715, the first-in-class cereblon modulator (CELMoD) for c-MYC protein (Nishida, ASH 2022). CyTOF confirmed the presence of much increased c-MYC protein levels in primary CD34+ AML than in CD34+ NBM hematopoietic stem cells (HSCs). Notably, CD34+ AML cells showed much greater sensitivity to GT19715 compared to CD34+ NBM cells. Data suggest on-target activity of GT19715 against c-MYC in LSCs with a therapeutic window between LSCs vs. NBM HSCs. Intriguingly, c-MYC protein levels are higher in CD34+CD38+ than in CD34+CD38- AML cells, suggesting that c-MYC drives the proliferation of AML progenitor cells differentiating from quiescent LSCs. Consequently, CD34+CD38+ AML progenitor cells exhibited greater sensitivity to GT19715 compared to CD34+CD38- LSCs. Using paired, TP53 null HL-60 GT19715-sensitive and -resistant cells generated through chronic exposure to GT19715, we interrogated the impact of GT19715 on metabolic changes. GT19715 induced pronounced reductions in basal and maximal oxygen consumption rates (OCRs) in GT19715-sensitive HL-60 cells. GT19715 reduced both basal, glutamine- and glucose-dependent extracellular acidification rates (ECARs) in GT19715-sensitive cells while no inhibition in OCRs or ECARs was observed in GT19715-resistant cells. GT19715 severely inhibited ECARs even after adding glucose to GT19715-sensitive cells, suggesting irreversible inhibition of glycolysis as one of the mechanisms of action of GT19715. GT19715 profoundly reduced AML blasts in TP53mut AML samples (N = 3) ( Fig. B), and in a very aggressive patient-derived xenograft (PDX) TP53mut AML model established from a patient with TP53 p.Y220C and p.P151A mutations along with MECOM rearrangement and K/NRAS mutations. In humanized Crbn I391V mice, where the Crbn-mediated protein degradation is operational, GT19715 only reduced WBC counts along with minimal body weight loss. GT19715 but did not reduce total mouse BM CD45+ cells, suggesting favorable toxicity profiles of GT19715. In conclusion, TP53mut AML comprised highly enriched LSC populations compared to TP53wt AML and targeting of c-MYC protein is highly effective in TP53mut AML in vitro and in vivo with a therapeutic window between AML LSC and normal hematopoietic cells.

Deregulation of HLA-Class I and Class II Genes in Chip, MDS, and Acute Leukemia and Similarity between Chip and MDS in HLA Profile

Introduction: Human leukocyte antigen (HLA) plays a major role in the interaction between the immune system and oncogenic process in various types of tumors. Emerging data suggests that the HLA system may have profound effects on response to immunotherapy and other types of therapy. Structural defects in HLA genes and variation in methylation have been reported to play a role in the expression of various HLA-I and HLA-II genes. Furthermore, an association between the expression of specific HLA-I or HLA-II genes and prognosis and outcome has been reported in some types of leukemia. Since HLA-I is expressed on all cells while HLA-II is expressed mainly in antigen presenting cells, we hypothesized that profiling the expression levels of both HLA-I and HLA-II in the bone marrow of patients with various myeloid neoplasms may provide information on the interaction between the microenvironment and the leukemic cell in these diseases. We used next generation sequencing to evaluate the expression level of HLA-I and HLA-II genes in myeloid neoplasms and compared these levels to normal bone marrow control. We used targeted next generation sequencing (NGS) for quantification of the RNA expression of 15 HLA-I and HLA-II genes. Methods: RNA was extracted from fresh bone marrow aspiration samples from 115 acute myeloid leukemia (AML), 196 clonal hematopoiesis of indeterminate potential (CHIP), 84 myeloproliferative neoplasm (MPN), 177 myelodysplastic syndrome (MDS), 40 acute lymphoblastic leukemia (ALL), and 279 normal bone marrow cases. RNA sequencing was performed using a targeted hybrid capture panel. The sequenced HLA-I genes were HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-H. The sequenced HLA-II genes were: HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DQB2, HLA-DRA, HLA-DRB1, HLA-DRB5, and HLA-DRB6. Salmon v1.4.0 software was used for expression quantification (TPM). To evaluate the depth of the significance in variation of the expression profiles between diseases we tested machine learning algorithm (random forest) incorporating the expression of all 15 genes in blindly classifying diseases. Two thirds of samples were used for training the random forest algorithm and one third was used for testing. Results: Bone marrow samples with CHIP showed no significant difference from normal bone marrow in level of expression of HLA-I genes but showed significantly (P <0.001) lower level of most HLA-II markers (DMB, DQA1, DQB2, DRB1, and DRB5). The same pattern was observed in MDS samples. In contrast, AML showed significant (P < 0.001) increase in the expression of both HLA-1 genes (A, C, and F) and HLA-II genes (DQA1, DPA1, DQB1, DRB6, and DPB1). Bone marrow samples from ALL patients showed significantly (P< 0.001) higher expression levels in DRB1 and DQA1 as compared with normal bone marrow. MPN samples showed no statistically significant difference from normal BM in any of the HLA-I or HLA-II genes, after adjusting for multiple testing. Machine leaning algorithm poorly classified MDS and AML and suggests complex overlap between the two diseases in HLA response. In contrast, the machine learning algorithm showed significant difference between MPN, MDS, and AML, and ability to distinguish all MPN cases with AUC of 0.738 (95% CI : 0.546-0.930) in the testing set (see figure). Conclusions: These findings show that the bone marrow microenvironment immune profile in MDS and CHIP likely plays a major role in the disease. Furthermore, this data suggests that this immune microenvironment is critical in CHIP and perhaps that therapeutic approaches supporting this immune microenvironment may help in preventing CHIP from progressing to MDS. The data also supports that AML is a disease that completely overwhelms the HLA immune system. In contrast, MPN appears to evade the HLA system and perhaps activating this system may help in the therapy of MPN.

Disease Monitoring in Acute Myeloid Leukemia with CLEC2A qRT-PCR

CLEC2A, C-Type Lectin Domain Family 2 Member A, is a newly discovered AML-restricted antigen. Detailed characteristics, clinical correlations, and therapeutic potential of CLEC2A in AML is presented by Kirkey et. Al. (ASH 2023). CLEC2A is entirely silent in normal hematopoiesis and uniquely expressed in patients with KMT2A rearrangements, providing an ideal therapeutic target in this high-risk subtype of AML. To date, CLEC2A has not been implicated in hematologic malignancies and expression of CLEC2A is entirely absent from normal tissues other than minimal expression in the skin. In addition to its utility as a therapeutic target, CLEC2A provides a unique target for disease monitoring given its lack of expression in normal hematopoietic cells. Here we present a highly sensitive quantitative molecular assay for CLEC2A, establishing its utility for disease monitoring in patients with KMT2A-r AML. In our efforts to evaluate CLEC2A expression and demonstrate the utility of using CLEC2A for disease monitoring in KMT2A-r, we developed a qRT-PCR assay using probes directed at exon 3 of the CLEC2A gene. The assay was optimized with RNA from the OCI-AML2 cells, validated to be CLEC2A-positive by flow cytometry and RNA from Kasumi-1 and K562 cells, both CLEC2A-negative by flow cytometry. We established the sensitivity of the assay through serial dilution of OCI-AML2 in a background of CLEC2A-negative Kasumi-1 or K562 cell RNA with concentrations ranging from 100% to 0.0001% OCI-AML2 RNA. The sensitivity assay demonstrated CLEC2A was detected at a level of 0.001% in both Kasumi-1 and K562. Given the association of CLEC2A with KMT2A-r AML, we evaluated the prevalence of CLEC2A as assessed by qRT-PCR. We screened diagnostic specimens from a large cohort of patients enrolled on recent COG Phase III clinical trials (ages 0-29) with and without KMT2A fusions to establish prevalence of CLEC2A expression. Using our CLEC2A qRT-PCR assay we evaluated diagnostic specimens from 122 patients with (N=76) and without (N=46) KMT2A-r, in addition to RNA from 8 normal bone marrow (NBM) samples, as controls and validated CLEC2A was entirely absent in NBM with median expression 0.13 copies per 1e 4 GUSB, figure 1A. Of evaluable samples, CLEC2A was detected in 65 of 76 (85%) KMT2A-r cases with a median expression of 317.45 copies per 1e 4 GUSB (range 0.03 - 21134.21 copies per 1e 4 GUSB). CLEC2A was detected in 21 of 46 (45%) non-KMT2A-r cases tested with a median expression of 1.37 copies per 1e 4 GUSB (range 0 - 1204.84 copies per 1e 4 GUSB). Further evaluation of KMT2A fusion partner showed that CLEC2A was detected in 100% of MLLT4 (N=6/6), 83.3% in MLLT10 (N=20/24), 72.7% of ELL (N=8/11), and 42.9% in MLLT3 (N=21/49). While CLEC2A expression was seen in both KMT2A-r and non-KMT2A-r AML patients, CLEC2A levels were significantly higher in KMT2A-r patients compared to non-KMT2A-r and NBM samples (p < 0.0001). Since CLEC2A is expressed in AML with no expression in normal hematopoietic cells, we hypothesized that monitoring CLEC2A via qRT-PCR throughout treatment (i.e., at diagnosis, at end of induction [EOI], at relapse) would provide added specificity to traditional flow cytometric disease monitoring. Using paired RNA samples from patients across multiple timepoints, we evaluated the change in CLEC2A expression throughout treatment, figure 1B. Patients with high CLEC2A expression at diagnosis (mean 1670 copies per 1e 4 GUSB, n=39) had decreased expression at EOI (mean 199 copies per 1e 4 GUSB, n=45) indicating treatment response, however high CLEC2A expression was seen at time of relapse (mean 1849 copies per 1e 4 GUSB, n=13) with CLEC2A expression preserved at relapse for all patients. qRT-PCR was able to detect CLEC2A expression in 7 patients at EOI who were in a morphologic CR with 6/7 (86%) patients being MRD-negative, further highlighting the increased sensitivity of CLEC2A qRT-PCR. Here we describe qRT-PCR monitoring of CLEC2A, a novel AML-restricted antigen highly enriched in KMT2A-r AML, provides added specificity and sensitivity to traditional flow cytometric MRD analysis. CLEC2A monitoring via qRT-PCR can be used to assess disease response throughout treatment and evaluate impending relapse as CLEC2A expression is retained at relapse. Additionally, qRT-PCR monitoring of CLEC2A can be incorporated in to pre- and post-HSCT disease evaluations similarly to qRT-PCR analysis of other known high-risk fusions such as NUP98-NSD1 fusions.

Unveiling Novel Therapeutic Targets for CAR Therapy in Multiple Myeloma through Single-Cell RNA Sequencing

Multiple Myeloma (MM) is a hematological malignancy characterized by the clonal proliferation of plasma cells. Among the different therapeutic options in this setting, chimeric antigen receptor (CAR) T cells recently showed unprecedent achievements in term of progression free survival. However, despite advancements in current therapies, the disease's heterogeneity remains a major challenge and all patients unavoidably relapse, rendering innovative approaches to identify precise therapeutic targets eagerly awaited. Along this line, the revolution of single-cell (sc) technologies brings new opportunities to identify precise therapeutic targets, including abundant and unique surface proteins for CAR therapy. In this study, by analyzing individual cancer cells at sc level, we aim to identify novel surface targets, facilitating the development of novel personalized cellular therapies as well as treatment sequences. To this end, we used samples collected from 9 publicly available single-cell RNA sequencing (scRNA-seq) datasets (GEO accession numbers: GSE176131, GSE189460, GSE223060, GSE210079, GSE145977, GSE124310, GSE161801, GSE163278, GSE161722) obtained from 156 patients affected by monoclonal gammopathies (19 monoclonal gammopathy of undetermined significance (MGUS), 10 smoldering multiple myeloma (SMM), 78 MM and 25 relapsed/refractory multiple myeloma (RRMM)) and 24 normal bone marrows (NBM), to conduct an unbiased search for genes showing specific expression in plasma cells (PCs), regardless of disease status. We found 15 genes showing differential expression in PCs (adj.pval<0.01 and logFC>1) and coding for surface proteins: TNFRSF17(BCMA), SDC1 (CD138), FCRL5 (GPRC5D), TNFRSF13B (TACI), CD38, SLAMF7 (CS1), CD59, FCGR2B, FGFR3, SLC44A1 (CTL1), CD320, FCRL2, IL15RA, INSR and SLAMF1. As expected, most of these molecules already represent critical therapeutic targets. After completing our initial analysis, we compared PCs transcriptomes from MM/RRMM patients with NBM, MGUS, and SMM samples. Among previously identified genes, CD320 only was significantly overexpressed in MM PCs. Next, we investigated associations between cytogenetic-related cell subclusters and previously identified surface markers. Interestingly, we found a significant association between the expression levels of TACI/CD59 and the overexpression of CCND2 and MAF, both associated with the presence of t(14:16). Next, we decided to focus on the first five genes (according to expression ranking) that do not currently have an active clinical development program in advanced phases, namely: TNFRSF13B, CD59, FCGR2B, SLC44A1, and CD320 (Fig. 1 A, values dichotomized based on their median) to uncover their (potential) impact/association with patient's outcome. In our analysis of different available expression profiling datasets (GSE4204, GSE2658, GSE57317, GSE4581, GSE4452, GSE9782, the CoMMpass study NCT01454297) involving around 2000 MM patients, we observed TNFRSF13B, CD59, and FCGR2B expression correlated with improved outcome while CD320 and SLC44A1 expression associated with worse outcome (Fig. 1 B). In conclusion, we integrated results from both scRNAseq and bulk-RNAseq/microarray data to identify new targets genes to be used in the context of personalized medicine. Interestingly, CD320 emerges as a potential candidate biomarker useful for monitoring disease evolution and predicting worse outcomes. Additionally, this approach led us to identify a series of potential new targets for MM patients, depicting a new scenario where each patient could be “screened” to identify the best molecule to be targeted. While further investigation is necessary to assess off-target toxicity and confirm clinical relevance, our analyses significantly streamline the search for tumor markers with a method potentially applicable to different malignancies, bringing us closer to identifying the best candidates for effective CAR therapy.

The Laip-Based-Dfn Approach Is Superior in Terms of Useful MRD Results As Compared to the Laip Approach after Cycle II in Acute Myeloid Leukemia

Clinical decision-making based on measurable residual disease (MRD) in acute myeloid leukemia (AML) is steadily increasing; therefore, accurate MRD results are imperative. Multi-parameter flow cytometry (MFC) is often used due to wide availability, particularly when no suitable mutation is present. Two approaches are most common in MFC-MRD: 1. Leukemia-associated immunophenotype (LAIP) approach, in which the aberrant phenotype is identified at diagnosis and tracked during follow-up. 2. Different-from-Normal (DfN) approach, in which aberrant surface antigen expression patterns not present in normal bone marrow (empty spaces in 2D dot plots) are identified at follow-up. The LAIP approach is sensitive, but does not consider immunophenotypic shifts and cannot be used when no LAIP is identified at diagnosis or when a diagnosis sample is unavailable. The DfN approach can detect these shifts, but ample expertise is needed to determine a malignant population. In the ELN-DAVID AML-MRD guidelines 2022, it is recommended to use the combination of the LAIP approach and the DfN approach to use the advantages of both approaches. In the HOVON-SAKK132 (HO132), the MRD was based on the combination of MFC-MRD and NPM1 RQ-PCR. Initially, the MFC-MRD in this study was based on the LAIP approach. However, the complete MFC panel (four 8-color tubes) published by Cloos et al. (J.Vis.Exp, 2018) was used for measuring MRD during follow-up. Therefore in this study, we had the unique opportunity to retrospectively analyze the samples after induction cycle 2 (C2) of patients in complete remission (CR/CRi) and determine additional MRD results with the LAIP-based-DfN approach. The original LAIP approach encompasses determining the percentages of the LAIPs in the follow-up sample, which were also identified at diagnosis. For the LAIP-based-DfN approach, we followed the same approach, but determined the percentages of all possible LAIPs in the MFC-MRD panel at diagnosis and after C2. An emerging LAIP is defined as a LAIP that is present in the follow-up but was not found at diagnosis. MRD positivity was defined as ≥ 0.1% LAIP cells of white blood cells. Patients with an emerging LAIP < 0.1% or no LAIPs in follow-up would result in MRD negativity for LAIP-based-DfN, but was only possible when at least 500,000 cells were acquired in all four tubes. At diagnosis, we received 884 samples of 905 patients (98%), in which we found a LAIP ≥ 10% of CD45dim blasts in 83% of the samples (Figure 1A). After C2, we received 615 samples, for which we had MFC-MRD results for 79% (487/615) by the LAIP approach. When we applied the LAIP-based-DfN approach, we scored 69 of 615 extra MRD results: an emerging LAIP ≥ 0.1% (MRDpos, N = 14), no LAIPs (MRDneg, N = 25) or an emerging LAIP < 0.1% (MRDneg, N = 30). When including these results in the data of the LAIP approach, the percentage of patients with an MRD result increased from 79% (487/615) to 90% (556/615), and the MRD positive percentage increased from 13% (81/615) to 19% (118/615)(Figure 1B). The LAIPs of emerging clones most often included CD56, CD15, and CD22. To assess the prognostic value in the HO132, we combined the reanalyzed LAIP-based-DfN MFC-MRD with the NPM1-MRD (N = 567). In these analyses, overall survival (OS) hazard ratio (HR) 95% confidence interval (CI) 1.92 (1.44-2.56) and relapse-free survival (RFS) HR 95% CI 1.7 (1.31-2.19) were similar to the original LAIP based/ NPM1 approach: OS HR: 95%CI 2.02 (1.48-2.76) and RFS HR: 95%CI 1.78 (1.34-2.35), respectively. In conclusion, the LAIP-based-DfN approach adds additional MRD results while retaining the prognostic value. Since HOVON-SAKK centers are highly compliant with the protocol and 98% of diagnosis samples were available, the LAIP-based-DfN approach can offer additional value for studies with limited access to diagnosis samples.

Polyamines Are Required for AML LSC Function through Their Role in Regulating eIF5A Dependent Protein Synthesis

Outcomes for patients with acute myeloid leukemia (AML) remain poor due to the inability of current therapies to fully eradicate disease initiating leukemia stem cells (LSCs) (Shlush et al., 2017). Many studies have demonstrated that LSCs have unique metabolic properties compared to AML blasts and normal hematopoietic stem and progenitor cells (HSPCs) (Jones et al., 2021). In this study we sought to quantify metabolite levels in LSCs compared to HSPCs using an unbiased metabolites screen. To examine metabolite levels in enriched LSCs and HSPCs we used mass spectrometry-based metabolomics analysis on enriched LSCs from 18 AML patients and HSPCs from 5 normal bone marrow (BM) specimens. Pathway analysis showed that arginine metabolism and biosynthesis were the most significantly enriched pathways in LSCs compared to HSPCs. Clinical studies have shown limited efficacy of targeting arginine levels using arginine degrading enzymes; therefore, we sought to target pathways downstream of arginine. To identify the pathways that arginine metabolism supports in LSCs, we used stable isotope labeled tracing analysis which showed arginine was metabolized through the urea cycle into polyamines in LSCs. Interestingly, the polyamine spermidine was the most significantly enriched metabolite in LSCs compared to HSPCs in our metabolomics analysis. Moreover, expression of SAT1, the enzyme catalyzing the export of polyamines from the cells, was decreased in AML compared to normal BM. Based on these observations, we sought to determine if the polyamine pathway represents a metabolic vulnerability that can be exploited to target LSCs. Polyamines are cationic compounds which have been previously shown to be important for the growth of various cancers but their role in AML LSCs has yet to be determined. To investigate if polyamines are essential for LSC survival and function, we treated LSCs and HSPCs with N 1, N 11-diethylnorspermine (DENSpm), a polyamine analog causing polyamine depletion through increased SAT1 expression and subsequent cellular export. DENSpm treatment decreased the viability of LSCs in 10 out of 12 of patients tested but had no impact on HSPCs viability. Further, treatment with DENSpm resulted in decreased engraftment potential of 5 AML specimens but did not alter the engraftment of HSPCs from 3 normal BM specimens, suggesting that targeting polyamines represents a promising approach to kill LSCs while sparing HSPCs. We then investigated the mechanisms by which polyamines are essential in LSCs. Using RNA-sequencing analysis on enriched LSCs from 5 AML patients and HSPCs from 3 normal BM specimens we observed an enrichment in myeloid differentiation signatures in DENSpm-treated LSCs but not in DENSpm-treated HSPCs. In line with this, expression of myeloid markers CD11b and CD15 were increased in patient-derived xenograft models engrafted with DENSpm-treated LSCs. As we did not observe such dysregulation in mice engrafted with DENSpm-treated HSPCs, these data suggest polyamine depletion promotes differentiation of LSCs but not HSPCs. Our transcriptomic analysis also unveiled decreased expression of genes involved in protein synthesis in DENSpm-treated LSCs but not in normal HSPCs. Protein synthesis and its regulation are critical for AML survival (Chen et al., 2019; Messling et al., 2022) but the role of polyamines in protein synthesis in LSCs has not been explored. Polyamines stimulate translation notably by serving as precursors for the hypusination of eukaryotic translation initiation factor 5A (eIF5A) (Park et al., 2010). Accordingly, DENSpm led to decreased hypusination of eIF5A in AML cells. This was associated with reduced protein synthesis level in AML cell lines and LSCs but not in normal HSPCs. Strikingly, eIF5A hypusination, protein synthesis and viability were rescued upon co-treatment with spermidine. Moreover, knockdown of eIF5A using siRNA resulted in impaired colony forming potential of AML and altered engraftment in patient-derived xenograft models. Altogether, these results suggest that DENSpm alters AML cell and LSC viability and function at least in part through decreased eIF5A-mediated protein translation. Altogether, our data demonstrates that depletion of polyamines decreased LSC function while sparing normal HSPCs. Mechanistically, our data suggest that LSC targeting is accomplished through decreased eIF5A-mediated synthesis of proteins crucial for LSCs.

Single Cell Multi-Omic Analysis of Tumor Microenvironment Evolution across the Disease Spectrum of Multiple Myeloma Identifies Differential Mechanisms of Immune Suppression

Background: Multiple myeloma (MM) is preceded by the precursor states of monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). The transformation of these precursors into myeloma is influenced not only by the evolution of tumor itself but also by the tumor microenvironment (TME). However, the specific molecular mechanisms and factors within the TME that drive this malignant progression remain largely unknown. Therefore, exploring the TME from non-malignant precursor stages to active myeloma could facilitate the development of novel therapeutic strategies aimed at slowing down this progression and possibly eradicating MM. Methods: We performed paired scRNA-seq, scTCR-seq and scBCR-seq on 148 freshly collected BM aspirate samplesincluding 73 relapsed refractory (RR) RRMM, 16 newly diagnosed (ND) MM, 34 SMM, 21 MGUS and 4 normal BM (nBM) samples. BM aspirates were subjected to CD138+ selection (n=19) or processed as whole BM (WBM) (n=129, among which 73 samples were also enriched for CD138+ cells). cDNA libraries were prepared from enriched CD138+ (n=92) and WBM samples (n=129) followed by simultaneous 5' gene expression (scRNA-seq) and T/B cell receptor sequencing (scTCR/BCR-seq). Single-cell data was processed as previously described (Dang et. al, Cancer Cell, 2023). Results: We profiled 656,371 high-quality cells including 344,665 CD138+ and 311,706 TME cells. We also obtained scTCR-seq and scBCR-seq data on 86,244 and 566,155 cells, respectively. Among them, 394,187 out of 652,399 cells had paired scRNA-seq data. Unsupervised clustering analysis revealed 15 different major TME cell types. Sub-clustering of each major cell type further resolved them into different subsets, resulting in a total of 94 TME cell subsets ( Figure). Overall, we observed a decreased proportion of CD4 T cells, B cells and progenitors and the enrichment of CD8 T cells, monocytes, and neutrophils along the nBM-MGUS-SMM-NDMM-RRMM axis. Notably, the most profound changes were identified within the context of RRMM. In lymphoid cells, we observed a stepwise decrease in mature B cells during disease progression. For T and NK cells, the naïve-like subsets were more abundant in normal bone marrow and the precursors. In contrast, the effector/cytotoxic subsets were enriched in symptomatic MM (NDMM and RRMM). Immunoregulatory T cells, including CD4T_C2 (Treg), CD4T_C4 (Th17) and CD4T_C7 (Tfh), showed an increased proportion within CD4 T cells in MM, particularly in RRMM compared with precursors. Furthermore, we identified a small exhaustion-like CD8 T cell subset which were enriched in RRMM. In myeloid cells, the classical monocytes showed a high degree of transcriptional homogeneity while the non-classical and intermediate monocytes were more heterogeneous. We observed a unique CD16_Mono_C0 subset which downregulated a considerable number of cytokine/chemokine receptors, almost exclusively unique to MM. The macrophage subsets displaying high phagocytosis signature (Macro_C0/3) were enriched in precursors. The monocyte-like macrophages (Macro_C1/2/4), which showed an angiogenesis phenotype, showed greater abundance in RRMM. Neutrophils were found exclusively in symptomatic MM, demonstrating elevated expression levels of TGFB1. Among the neutrophil subsets, those expressing myeloid checkpoints such as PILRA, SIRPA, and VSIR (Neutro_C0/3/4) exhibited increased prevalence in RRMM. In contrast, subsets expressing ARG1 and MMP9 (Neutro_C1/2) were more enriched in NDMM. Within the dendritic cell population, a specific subset characterized by high TGFB1 and VEGFA expression (cDC2_C1) was exclusively present in MM. Additionally, we observed an enriched proliferative dendritic cell subset in MM. Conclusions: We observed a notable pattern of cellular changes along the nBM-MGUS-SMM-NDMM-RRMM axis, indicating a progressive increase in immune suppression primarily driven by myeloid cells. These observations provide valuable insights into immune cell dynamics, their functional characteristics, and potential roles during disease progression and highlight potential targets for further investigation, especially in the context of RRMM.

CD276 (B7-H3) Is an Immunotherapeutic Target in Acute Myeloid Leukemia with Preclinical Efficacy of Vobramitamab Duocarmazine, an Investigational CD276 Antibody-Drug Conjugate

CD276 (B7-H3) is an immune checkpoint inhibitor expressed in a majority of solid tumors and hematologic malignancies, however it has not been well-described in acute myeloid leukemia (AML). A variety of immunotherapies targeting CD276 including CAR T cells and antibody-drug conjugates (ADCs) are being explored in solid tumors, however their role in leukemia has not been well elucidated. We interrogated the transcriptome and associated clinical and laboratory data from a large cohort of pediatric and adult AML patients to define the prevalence, frequency of co-occurring genetic alterations and outcomes associated with CD276 expression. Lastly, we evaluated preclinical in vitro efficacy in AML of vobramitamab duocarmazine (vobra duo), an investigational ADC with a humanized B7-H3 mAb conjugated via a cleavable linker to the prodrug seco-DUocarmycin hydroxyBenzamide Azaindole. Ribo-depleted RNA sequencing data from 2,241 patients with AML (Pediatric ages 0-29 years N=1,412, from recent Phase 3 COG clinical trials; Adult ages 18-88 years BEAT AML N=451, SWOG N=199, TCGA-LAML N=179) with available cytogenetic, molecular, and clinical data were utilized. Conventional karyotyping and mutational analysis including NGS were used to determine additional cytomolecular aberrations. Response was measured by 5-year overall survival (OS), event-free survival (EFS) and relapse risk (RR). Of the 1,412 pediatric patients evaluated, CD276 mRNA expression was positive (defined as mRNA transcripts per million (TPM) >2) in 289 patients (20%). Higher prevalence (58%) was seen in adult AML patients (BEAT AML N=209/451, SWOG N=191/199, TCGA-LAML N=85/179), Fig 1A. Flow cytometry validated cell surface expression in pediatric AML, with 22% of patients expressing CD276. Cell surface expression was also observed in AML cell lines. CD276 mRNA expression is absent in normal hematopoietic cells (TPM <1) with absent cell surface expression in normal bone marrow as assessed by flow cytometry, allowing for targeted CD276 therapies to be utilized with no/minimal on-target off-tumor hematopoietic toxicity. Evaluation of co-occurring cytomolecular aberrations demonstrated a significant enrichment of CD276+ pediatric patients with high-risk genetic alterations, most notably KMT2A fusions (N=150/343, 43.7%), KAT6A-CREBBP (N=11/12, 91.6%) and CBFA2T3-GLIS2 (N=22/39, 56.4%). We evaluated the significance of CD276 mRNA expression on clinical outcomes in childhood AML. 5 year event free survival (EFS) for patients with CD276 expression was 35% compared to 44% for patients without CD276 expression (p=0.0022). Overall survival (OS) was 52% for patients with CD276 expression compared to 61% for patients without CD276 expression (p=0.0015). Relapse was seen in 40.5% of patients with CD276 expression compared to 35.8% of patients without CD276 expression and relapse occurred on average earlier in CD276 positive patients (0.89 years vs. 1.06 years, p=0.02). Given the poor clinical outcomes for patients with high CD276 expression, novel targeted therapies are needed for this high risk cohort. With multiple CD276 immunotherapies under clinical evaluation in solid tumors, we tested vobra duo in OCI-AML2, a CD276 positive KMT2A-rearranged AML cell line, which demonstrated robust cytolytic activity with an IC50 less than 1nM (IC50=0.9306 nM) supporting the potential efficacy of CD276 targeted therapies in AML (Fig 1B). In a large cohort of AML patients, we identified CD276 to be overexpressed in 20% of pediatric AML patients and 58% of adult AML patients with a significant enrichment in high risk fusion subtypes including KMT2A-rearranged AML, KAT6A-CREBBP and CBFA2T3-GLIS2 fusions. Patients with CD276 expression have poor outcomes with significantly worse EFS and OS compared to patients who do not have CD276 expression. With multiple CD276 immunotherapies under clinical evaluation for solid tumors, we hypothesized that targeting CD276 in AML would result in specific cytolytic activity. Vobra duo showed robust in vitro cytolytic activity against CD276 positive AML cells highlighting the need for ongoing preclinical evaluations of CD276 targeted therapies in AML. Given the established safety profile for vobra duo this provides a clear path for rapid translation to clinical use for high risk AML patients.

CLEC2A Is a Novel AML-Restricted Immunotherapeutic Target Enriched in KMT2A-Rearranged Acute Myeloid Leukemia

KMT2A rearrangements (KMT2A-r) are a frequent event in childhood acute myeloid leukemia (AML) and in treatment related AML in adults leading to a highly refractory and deadly subtype of AML regardless of age. Targeted therapies for KMT2A-r AML have had limited success with novel therapies urgently needed. Despite successes in other leukemias, immunotherapy in AML is in its infancy in part due to significant overlap of antigens expressed in AML and normal hematopoietic cells, which would limit efficacy and increase hematopoietic toxicity. We interrogated the AML transcriptome compared to normal hematopoiesis to identify optimal targets that are silent in normal hematopoieisis and are highly expressed in AML. Here we describe a novel immunotherapeutic target, CLEC2A, a member of the C-type lectin family, that is highly enriched in high-risk AML, particularly KMT2A-r AML and absent in normal hematopoeisis. We evaluated the association between KMT2A fusions and CLEC2A expression identifying CLEC2A as a putative oncoprotein and the potential for CLEC2A to serve as an immunotherapeutic target in high-risk AML via antibody-mediated cytotoxicity. Diagnostic specimens from 1,864 patients with AML (Pediatric 0-29 years from COG Phase III trials N=1486; Adult 18-88 years SWOG N=199 and TCGA-LAML N=179) were interrogated using contemporary NGS to identify genetic aberrations. Conventional karyotyping and mutational analysis were used to determine additional cytogenetic and molecular abnormalities. As CLEC2A was entirely absent in normal bone marrow (TPM 0-0.15), positive CLEC2A expression was defined as mRNA transcripts per million (TPM) >1. Although, CLEC2A was detected in only 18% of AML patients (252/1486; 17% pediatric and 90/378; 24% adult patients), it was highly enriched in those with KMT2A-r AML with 77% (N=193) of CLEC2A positive patients having KMT2A-r, accounting for 48% of the total KMT2A-r cohort, Fig 1A. CLEC2A expression was enriched in specific fusion subgroups: MLLT10 (80%), MLLT4 (71%), and MLLT3 (48%), Fig 1B. CLEC2A expression was low to absent in a majority of other patients, though was seen in other high-risk groups including non-KMT2A MLLT10 fusions, NUP98 fusions, CBFA2T3-GLIS2 fusions, and monosomy 7. CLEC2A is also expressed in a subtype of T-ALL (HOXA activated), which is a fusion driven T-ALL. We further evaluated a potential causal link between KMT2A fusions and CLEC2A expression and inquired whether KMT2A fusion proteins might directly bind to the CLEC2A promoter and induce CLEC2A expression. We used AutoCUT&RUN profiling of genome-wide occupancy of KMT2A oncoproteins to evaluate direct binding of the KMT2A fusion protein to the CLEC2A promoter in primary AML patient samples with three different KMT2A fusions (MLLT10, MLLT4 and MLLT3). We found direct binding of KMT2A fusion proteins to the CLEC2A promoter region, indicating a causal relationship between KMT2A fusions and CLEC2A expression. We then evaluated the potential of therapeutic targeting of CLEC2A by an antibody-drug conjugate using a HumZap cytotoxicy assay. CLEC2A antibody conjugated to saporin (a toxin with cytolytic activity when internalized) was incubated with CLEC2A+ and CLEC2A- cells with target-specific cytotoxicity evaluated by colorimetric assay. CLEC2A+ OCI-AML2 cells had significantly higher rate of cytotoxicity compared to CLEC2A- control cells at 10nM Ab-toxin conjugate. Clinical outcomes for patients with and without CLEC2A expression was evaluated. Patients with CLEC2A had significantly worse outcomes (EFS 23% vs. 45.5%, p<0.0001 and OS 38% vs. 63%, p<0.0001 for patients with and without CLEC2A expression, respectively). Given high rates of co-occurring KMT2A-r AML, particularly high-risk fusions (MLLT10 and MLLT4), we evaluated clinical outcomes within the KMT2A-r cohort as these patients have poor EFS and OS. KMT2A-r patients with CLEC2A had significantly worse EFS (24% vs. 39%, p=0.0063) and OS (38% vs. 60%, p=0.0002). Within the KMT2A-r cohort, high rates of relapse were seen for patients with CLEC2A expression compared to those without CLEC2A (72% vs. 55%, p=0.0039). Here we describe CLEC2A, a novel AML-restricted cell surface target that is an ideal immunotherapeutic target. CLEC2A is highly expressed in KMT2A-r AML, entirely absent in normal hematopoietic cells, directly and causally linked to the KMT2A fusion, and can be used for target-directed cytotoxicity.

Single-Cell Genomics Details the Maturation Block in BCP-ALL and Identifies Therapeutic Vulnerabilities in DUX4-Rearranged Cases

B cell progenitor acute lymphoblastic leukemia (BCP-ALL) is the most common childhood malignancy. It is initiated by multiple genetic alterations, causing a maturation arrest and accumulation of abnormal progenitor B cells. Current treatment protocols with chemotherapy have led to favorable outcomes, but are associated with significant toxicity and risk of side effects, highlighting the necessity for highly effective, less toxic, targeted drugs that also show efficacy in children experiencing relapse. We have used multimodal single-cell sequencing to delineate the transcriptional, epigenetic, and immunophenotypic characteristics of 23 childhood BCP-ALLs, including the BCR::ABL1-positive, ETV6::RUNX1-positive, high hyperdiploid, and recently discovered DUX4-rearranged subtypes. In total, 188,546 single cells derived from 23 BCP-ALLs and 9 normal bone marrow samples were profiled using the 10X Genomics platform. Cellranger multi and atac count (10X Genomics) were used to generate read count matrices. Further analysis was performed using Seurat and Signac. The in-house developed program SingleCellProjections was used to create a normal bone marrow reference graph onto which the BCP-ALL data was projected. Projection of the ALL cells along the normal B cell differentiation axis, revealed a diversity in the maturation block between the different BCP-ALL subtypes. Whereas the BCR::ABL1-, ETV6::RUNX1-positive and high hyperdiploidy cells mainly showed similarities to normal pro-B cells, the DUX4-rearranged ALL cells also displayed a transcriptional signature resembling mature B cells. In addition, the blast population in DUX4-rearranged ALLs showed multilineage priming toward non-hematopoietic cells, myeloid and T cell lineages, but also an activation of PI3K/AKT signaling that sensitized the cells to PI3K inhibition. Finally, we show that chimeric antigen receptor T cell therapy targeting the upregulated myeloid receptor CD371 (CLL-1), effectively eliminates DUX4-rearranged ALL cells. Our results provide a detailed characterization of BCP-ALL at the single-cell level and reveal therapeutic vulnerabilities in the DUX4-rearranged subtype with important implications for the understanding of ALL biology and new therapeutic strategies.

Determination of Reference Values of Hematopoetic CELLS in Normal BONE Marrow By 10 Colors FLOW Cytometry in Brazilian Population

Purpose: To determine relative and absolute reference values of normality in individuals with no hematologic disease bone marrow, as well as normal patterns of maturation for the different hematopoietic lineages. Methods: Bone marrow samples were collected from 46 healthy patients (16 women and 30 men) undergoing orthopedic surgery and were aged between 18 and 87 years (median 36 years). The analysis of 8 tubes performed in an equipment with 10 colors configuration enabled to evaluate and quantify (% and mm³) B lymphocytes (immature and mature), T lymphocytes (helper, cytotoxic, double negative, double positive), natural killer cells, granulocytes (myeloblasts, promyelocytes, myelocytes, metamyelocytes, rods and neutrophils), monocytes (monoblasts, promonocytes, mature monocytes, classical, intermediate and non-classical), erythroblasts (immature and mature), plasma cells, basophils, mast cells, myeloid dendritic cells and plasmacytoid dendritic cells. The results were evaluated according to sex (female and male), age group (<40, 40 to 60, >60 years), and described as median percentage and absolute values. Results: In evaluating the results according to age group, we identified that older individuals presented an increase in plasma cells that expressed CD56 (p=0.031 in absolute values). In addition, they also had increase relation of CD4/CD8 ratio (p=0.002), mast cells (p=0.004 in percentages), non-classical monocytes (p=0.016 in percentages), as well as a decrease in cytotoxic T lymphocytes (p=0.031 in absolute values), double negative T lymphocytes (p=0.018 in absolute values), and natural killer cells that express CD56 (p =0.002 in absolute values and p=0.012 in percentages). Moreover, these individuals also presented higher natural killer cells that expressed CD56 and CD16 (p=0.024 in absolute values), myeloblasts (p=0.048 in absolute values), metamyelocytes/Rods (p=0.038 in absolute values), neutrophils (p=0.006 in absolute values), monoblasts (p=0.030 in absolute values), mature monocytes (p=0.024 in absolute values), classical monocytes (p=0.017), plasmacytoid dendritic cells (p=0.027 in absolute values), and mature erythroblasts (p=0.004 in absolute values). In the evaluation of the results according to sex, we identified that the population of mature B lymphocytes (p=0.013) and B lymphocytes that expressed CD5 (p=0.014) was higher in women, while the percentage of natural killer cells was relatively higher than in men (p=0.007), particularly in the population of cells that expressed CD56 and CD16 (p=0.013). Conclusion: This study established reference values (absolute and relative) of B lymphocytes (immature and mature), T lymphocytes (helper, cytotoxic, double negative, double positive), natural killer cells, granulocytes (myeloblasts, promyelocytes, myelocytes, metamyelocytes, rods and neutrophils), monocytes (monoblasts, promonocytes, mature monocytes, classical, intermediate and non-classical), erythroblasts (immature and mature), plasma cells, basophils, mast cells, myeloid dendritic cells and plasmacytoid dendritic cells according sex and age group in bone marrow samples from individuals with no hematologic disease Maturational stages of B lymphocytes, granulocytes, monocytes and erythroblasts was shown to be stable regardless of sex and age.

Early Generation Telomerase Deficient Mice to Investigate Clonal Hematopoiesis in Aging

Clonal hematopoiesis is an age-associated condition characterized by the expansion of hematopoietic stem cell (HSC) pools with somatic mutations, observed in 10-30% of individuals older than 70 years. This phenomenon is primarily driven by mutations in transcriptional regulators such as DNMT3A, TET2, and ASXL1. While the clonal expansion of mutated HSCs is ubiquitous with age, it appears largely benign in the absence of strong selective pressures. These benign clones might compensate for the loss of regenerative HSC capacity during aging, and skew differentiation towards the myeloid lineage. Telomere erosion plays a significant role in this process. Telomeres normally prevent the activation of the DNA damage response (DDR) machinery. However, with each cell division, telomeres shorten until they reach a critical length, eventually leading to apoptosis or senescence. Previous studies have suggested a potential involvement of telomere attrition in the development of clonal hematopoiesis (Hinds et al. Blood 2016; Zink et al. Blood 2017). Late generation telomerase deficient (Terc-/- G5) mice develop premature bone marrow failure and mortality, however earlier generation Terc-/- G3 (3 homozygous crosses) have shortened telomeres but appear relatively normal. Importantly, inhibitors of telomerase have recently entered clinical trials for haematological malignancies and therefore understanding the long-term effects of telomerase inhibition is important. By studying the interplay between telomere attrition, HSC aging, and the development of CHIP, we aim to gain insights into the mechanisms underlying age-associated hematopoietic changes and their potential clinical implications. We examined young (age 8-10 week) and aged (10-12 month) Terc-/- G3 mice. Telomere lengths were significantly reduced in bone marrow cells from young Terc G3 (15 kb) when compared to wild-type mice (30 kb) with mild reduction in BM cellularity. Absolute long-term HSC numbers were reduced, as were megakaryocyte erythroid progenitors, but there was preservation of granulocyte macrophage progenitors and common myeloid progenitors. Spleen progenitor numbers were not affected. In comparison, aged (10-12 months old) Terc G3 mice showed enhanced differentiation skewing in the peripheral blood with significantly increased myeloid (n = 8 wt; n = 12 Terc ko; p = 0.0004) and concomitantly reduced B cell (p = 0.0002) and T cell (p = 0.0320) populations. There was marked mobilization of bone marrow progenitors to the spleen, most notably GMP (p = 0.0004) but also MEP and CMP in aged Terc G3. Furthermore, aged Terc G3 ko now exhibited marked reduction in bone marrow CMP (p < 0.0001) and MEP (p < 0.0001), but skewing towards GMP (p<0.001) resulting in normal total GMP numbers, but markedly reduced CMP and MEP numbers (p < 0.0001). Compared to young Terc G3 mice, the aged Terc G3 mice had relative expansion of the BM LT-HSC population, and therefore normal numbers of BM LT-HSC compared to aged matched controls. Aged Terc G3 BM LTHSC have been isolated for gene expression and clonality analysis. Together, these findings are consistent with accelerated aging in G3 Terc-/- mice, providing a model for examining clonal hematopoiesis. In order to assess the cell intrinsic function of Terc G3 LTHSC, we next performed non-competitive bone marrow transplantation experiments from Terc ko or wild-type donors into lethally irradiated young wild-type recipients (n = 9 per group), and analysed these at 4 months post-transplant. There was reduced, but stable reconstitution by Terc G3 cells, consistent with previous competitive bone marrow transplants in Terc G3 (Bruedigam et al. Cell Stem Cell 2014). We observed a similar reduction in bone marrow cellularity in Terc ko transplanted recipients (p = 0.003). Lineage skewing was less pronounced but CMP percentages were significantly reduced in Terc ko compared to wild-type conditions (p = 0.03). In contrast to primary mice, mobilization of progenitors to the spleen was not observed, suggesting that this finding was ameliorated by normal bone marrow and splenic supporting cells. The aged G3 Terc ko model faithfully recapitulates the haematopoietic phenotype of aging in humans and we propose to utilise this model to further our understanding of the mechanisms and physiological consequences of clonal hematopoiesis, to eventually help identify strategies to mitigate its clinical consequences.

Transposable Elements Are Differentially Expressed in MDS Stem Cells in a Disease-Stage-Specific Manner

Introduction Transposable elements (TEs) are widely spread across the human genome. Although TEs have been implicated in the pathogenesis of hematological malignancies, their expression in myelodysplastic syndromes (MDS) has not yet received extensive analysis. In the present study, we performed RNA sequence analysis of bone marrow stem and progenitor cells from patients with MDS and secondary acute myeloid leukemia (AML) and profiled differentially expressed TEs. Methods Bone marrow samples were obtained from each patient at the time of diagnosis. Normal bone marrow (NBM) samples from four healthy individuals were purchased from Lonza and processed in the same manner. After separation of CD34 + cells using magnetic beads, we purified lineage marker-negative (Lin -) CD34 + CD38 - stem cells and Lin - CD34 + CD38 + progenitor cells by cell sorting. The sorted cells were subjected to RNA-seq analysis using oligo-dT primers, which can capture poly-adenylated TE transcripts. Each read was aligned to the reference genome, then the annotation of the transcripts was performed using RepeatMasker, a program that screens DNA sequences for interspersed repeats and low-complexity DNA sequences (https://www.repeatmasker.org). Read counts for individual TEs were aggregated to the level of TE class, family, and subfamily according to hierarchies defined by the human RepeatMasker (repClass, repFamily, and repName, respectively). To generate raw counts of individual TE regions in RepeatMasker, the bedtools map function (version 2.27.1) was used with bed files of each sample. Normalization and significant expression differences were detected using DESeq2 (version 2.2.1). Differentially expressed TEs were defined using the cutoff of adjusted P values (q < 0.05). DESeq2 normalized counts were obtained from read counts by considering sequence depth, gene length, and RNA composition. Normalized counts were z-score scaled, and then subjected to uniform manifold approximation and projection (UMAP). Results Thirty-two patients were included in the present study. According to the 2016 revision to the World Health Organization classification (Arber DA, et al. Blood. 2016; 127: 2391-2405), 13 patients (40.6%) were diagnosed with MDS with multilineage dysplasia (MDS-MLD), 15 patients (46.9%) with MDS with excess blasts (MDS-EB), and 4 patients (12.5%) with AML with myelodysplasia-related changes (AML-MRC). We found that 26 and 17 repName TEs were significantly up-regulated in stem and progenitor cells compared with their NBM counterparts, and the majority of them belonged to LTRs, followed by DNA transposons. Since each LTR has multiple individual regions where similar sequences are scattered, LTR expression was further distinguished by the genomic locations of individual LTRs. Comparison of MDS-MLD, MDS-EB, and AML/MRC stem and progenitor cells to NBM counterparts revealed that more LTRs were differentially expressed in stem cells than in progenitor cells. Among them, many of the up-regulated LTRs were shared among different disease types (MDS-MLD, MDS-EB, and AML-MRC) in both stem and progenitor cells. AML-MRC had the largest number of differentially up- and down-regulated LTRs in stem cells. MDS-EB had the largest number of differentially up-regulated LTRs in progenitor cells, while only a small portion of LTRs were down-regulated in progenitor cells. LTRs at individual regions were differentially expressed in a more disease-specific manner, particularly in stem cells. Accordingly, UMAP plots that visualized the expression profiles of LTRs at individual regions showed that AML-MRC stem cells form a unique cluster, discriminating AML-MRC from MDS. To further understand the transcriptional reprogramming of LTRs during the disease progression from MDS to secondary AML, we analyzed the expression of differentially expressed LTRs at individual regions specific to AML-MRC by K-means clustering. They were subdivided into four clusters, of which Clusters 1, 3, and 4 showed gradual upregulation during disease progression, while Cluster 2 showed comparable upregulation at MDS and AML-MRC stages. Conclusions Our results suggest that specific LTRs behave in a disease-status and cell-type-specific manner in MDS and during leukemic transformation, providing fundamental information for understanding the pathogenesis of MDS.

Detection and Prevention of ADAR1p150-Induced Hematopoietic Stem and Progenitor Cell Aging

Introduction: Over the last decade, ADAR1p150 has been linked by our research team and others to malignant progenitor reprogramming and therapeutic resistance in a broad array of malignancies. However, ADAR1p150's role in accelerated hematopoietic stem and progenitor cell (HSPC) aging in response to inflammatory cytokines had not been clearly elucidated. Here, we investigate ADAR1 splice isoform switching and the ADAR1p150 inhibitory effects of Rebecsinib on human HSPC function in normal humanized aged normal bone marrow (aNBM) HSPC mouse models. Methods: Whole transcriptome RNA sequencing (RNA-seq) was utilized with splice isoform and editome analysis to detect ADAR1 splice isoform switching and adenosine to inosine RNA editing. Rebecsinib (17S-FD-895), a pre-IND drug candidate, has shown promising potential as an inhibitor of ADAR1p150-mediated RNA editing ( Crews, Ma...Jamieson. Cell Stem Cell 2023), which is an important therapeutic target in patients with myelofibrosis or acute myeloid leukemia that overexpress ADAR1p150 compared with ADAR1p110. Human CD34 + cells, isolated from aged normal bone marrow (aNBM) from patients undergoing elective hip replacement surgery, were transduced with a lentiviral ADAR1-nano-luciferase reporter and transplanted into NSG-SGM3 adult mice that secrete human SCF, IL-3, and GM-CSF cytokines that support normal hematopoiesis. After engraftment confirmation peripheral blood human CD45 flow cytometric assessment and IVIS 200 system imaging of ADAR1-nano-luc-GFP reporter expressing HSPC engraftment, the mice were randomly divided into vehicle-treated and Rebecsinib-treated groups (15mg/kg, twice a week for two weeks). Peripheral blood, bone marrow, and spleen were collected upon treatment completion for analysis. Results: By HSPC RNA-seq, we detected an imbalance between ADAR1p150 and ADAR1p110 expression together with increased RNA editing and splicing alterations using whole transcriptome RNA sequencing (RNA-seq) of aged compared with young bone marrow hematopoietic stem and progenitor cells (HSPCs). However, the functional relevance of ADAR1 splice isoform switching had not been evaluated in human-aged normal bone marrow HSPC xenograft (PDX) mouse models nor had this cytokine-induced HSPC aging been reversed. Flow cytometric analysis and immunohistochemistry staining revealed that Rebecsinib treatment at a dose of 10mg/kg, twice a week for two weeks, effectively spared normal human immune cells (CD3+, CD14+, and CD19+ cells). Remarkably, Rebecsinib treatment not only preserved Hematopoietic Stem (CD34+CD38-) cells and Progenitor (CD34+CD38+) cells in peripheral blood, bone marrow, and spleen but also promoted their expansion. These findings demonstrate the potential of Rebecsinib to enhance the retention of HSPC populations in the bone marrow niche. Conclusions: Our pre-clinical experiments utilizing RNA-seq, a novel ADAR1-nano-luciferase-GFP reporter, and aNBM PDX mouse models provide compelling evidence supporting the potential of Rebecsinib to restore the balance of ADAR1p150:p110 ratios and engraftment in humanized aNBM HSPC mouse models. These findings highlight Rebecsinib as a promising therapeutic candidate for targeting leukemia and myelofibrosis while preserving essential HSPC populations.

Dynamic Oscillation of Tetraspanin CD9 Orchestrates Engraftment and Mobilization of Human Hematopoietic Stem/Progenitor Cells

Background Trafficking of hematopoietic stem/progenitor cells (HSPCs) to and from their bone marrow (BM) niche is pivotal for engraftment and mobilization during clinical transplantation, where both processes are controlled by highly orchestrated molecular programs. We previously identified the tetraspanin CD9 as a downstream effector of the SDF-1/CXCR4 axis driving migration, adhesion and homing of human CD34+ HSPCs, but its consequential impact on stem cell engraftment and mobilization as well as the underlying mechanisms remains unknown. Methods Umbilical cord blood (UCB) CD34+ cells were exposed to a CD9 neutralizing antibody ALB6 prior to infusion into sublethally irradiated NOD/SCID mice, with overall engraftment and lineage output assessed by enumerating human CD45+ (pan-leukocytes), CD19+ (B-lymphoid), CD33+ (myeloid) and CD34+ (stem/progenitors) cells in the recipient BM at 8 weeks post-transplantation. CD9 expression on engrafted HSPCs was characterized by flow cytometry and compared to those before transplantation or after G-CSF mobilization. The oscillation pattern was then validated on HSPCs in pairwise BM, peripheral blood (PB) and G-CSF-mobilized peripheral blood (MPB) samples from healthy donors. Activity of integrin VLA-4 (an established binding partner of CD9) was evaluated on BM-residing and mobilized HSPCs by VCAM-1 binding assay. Results A short exposure of UCB CD34+ cells to the CD9 neutralizing antibody markedly inhibited their BM engraftment by 64.2% ( P=0.003), reflected by the general decline in human CD45+ cell population. There was no bias in lymphoid/myeloid lineage outputs, suggesting CD9-mediated engraftment is not restricted to specific subsets of hematopoietic precursors. CD9 expression was drastically elevated on engrafted CD34+ cells in the BM from 9.6% to 91.4% ( P<0.001) compared to pre-transplantation. In contrast, CD9 expression was significantly downregulated on BM CD34+ cells from 73.4% to 33.5% ( P<0.001) upon G-CSF mobilization, with the decline observed in both primitive stem cells (CD34+CD38-) and committed progenitors (CD34+CD38+), indicating that the oscillation of CD9 is highly associated with HSPC trafficking. Concordantly, pairwise analysis of steady-state HSPCs in BM and PB from healthy donors revealed higher CD9 level in BM-residing CD34+ cells than the PB-circulating portion (46.1% vs. 26.5%, P=0.004; n=13). In addition, a significantly higher CD9 expression was observed on BM CD34+ cells (47.7%; n=16) than those in MPB (32.3%, n=25; P=0.003). CD9 expression on MPB CD34+ cells was enhanced upon SDF-1 exposure ( P=0.006), revealing a dynamic regulation. In normal BM, CD9+ HSPCs exhibited elevated VLA-4 activities with preferential binding to soluble VCAM-1 compared to the CD9- counterpart (51.4% vs. 25.9%, P<0.001; n=10). Superiority in VCAM-1 binding capacity was also witnessed in BM-residing CD34+ cells compared to MPB (37.7% vs. 4.1%, P=0.014), implicating that the influence of CD9 on HSPC trafficking may due to the alteration of integrin activities and downstream adhesive properties. Conclusion This study describes the dynamic regulation of CD9 during entry and exit of HSPCs to and from their BM niche via modulation of integrin activities. The findings extend our understandings on HSPC trafficking that could potentially lead to new strategies to enhance clinical stem cell transplantation and mobilization.

Newly Identified Roles for PIEZO1 Mechanosensor in Controlling Normal Megakaryocyte Development and in Primary Myelofibrosis

Introduction: Mechanisms through which mature megakaryocytes (Mks) and their progenitors sense the bone marrow extracellular matrix (ECM) to promote lineage differentiation in health and disease are still partially understood. We found PIEZO1, a mechanosensitive cation channel, to be expressed in Mks. Human mutations in PIEZO1 have been described to be associated with blood cell disorders. Primary Myelofibrosis (PMF) is a Chromosome Philadelphia-negative Myeloproliferative Neoplasm (MPN), characterized by altered stiffer ECM and megakaryocytosis, often driven by the gain-of-function mutation in JAK2 V617F. Here, we study a role for PIEZO1 in megakaryopoiesis and proplatelet formation under normal physiological conditions and in the context of PMF. Methods: Bone marrow Mks from C57BL/6J control and myelofibrotic mice carrying the human JAK2 V617F+ mutation, or Mks derived from stem cells of patients carrying the same mutation were analyzed at mRNA and protein levels for Piezo1 expression. Effects of Piezo1 activation or inhibition on Mk maturation and ploidy were assessed by flow cytometry, and proplatelet formation by fluorescent microscopy. Human peripheral blood samples from PMF patients, diagnosed according to established criteria, or healthy individuals were used to compare the two categories for Piezo1 relative expression on mRNA and protein levels and platelet biogenesis. Mk-specific double knockout mice of Piezo1/2 were generated to compare platelet counts under normal conditions or bone marrow ablation with 5-FU challenge. Culture of mouse and human Mks in different 3D microenvironments was used to demonstrate in vitro association of Piezo1 expression levels with different ECM stiffness substrates. Results: Mouse Mks express PIEZO1 and negligible levels of PIEZO2. Pharmacological activation of mouse PIEZO1 increases the number of immature CD41 + Mks, while the number of mature CD41 +CD42 + Mks and Mk ploidy level are reduced. Piezo1/2 knockout mice show an increase in Mk size and platelet count, both at basal state and upon marrow regeneration. Similarly, in human samples, PIEZO1 is expressed during megakaryopoiesis. Its pharmacological activation reduces Mk size, ploidy, maturation, and proplatelet development. Resulting effects of PIEZO1 activation on Mks resemble the profile in PMF. Intriguingly, Mks derived from PMF mice bearing the Jak2 V617F mutation show significantly elevated PIEZO1 expression, compared to wild-type controls. Pharmacological activation of PIEZO1 in these cells significantly augments the number of immature CD41 + Mks, and sharply reduces the number of mature CD41 +CD42 + cells and ploidy level. Accordingly, Mks isolated from bone marrow aspirates of JAK2 V617FPMF patients show increased PIEZO1 expression compared to Essential Thrombocythemia. Additionally, PIEZO1 was overexpressed in 3D culture of human and mouse Mks in stiffer microenvironments as compared to softer ones and liquid medium. Most importantly, PIEZO1 expression in bone marrow Mks is inversely correlated with patient platelet count. The ploidy, maturation, and proplatelet formation of Mks from JAK2 V617FPMF patients are rescued upon PIEZO1 inhibition with GsMTx4. Discussion: Our data show that PIEZO1 might serve as a break of Mk maturation and platelet formation in physiology, and its upregulation in PMF Mks might contribute to aggravating some hallmarks of the disease. Our finding that inhibition of PIEZO1 activity partially rescues the aberrant Mk phenotype associated with PMF suggests that GsMTx4 holds potential as a therapeutic strategy to mitigate the pathological effects observed in Mk development in PMF patients.

Single-Cell Multiomics Analysis Reveals Potential Drivers of Response to the Anti-TIM3 Inhibitor Sabatolimab Combined with Azacitidine in MDS and CMML

Background The median survival for patients with newly diagnosed (ND) higher risk myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) remains poor. The potential curability of MDS and CMML with allogeneic hematopoietic stem cell transplantation supports the concept of anti-tumor immunity and has led to interest in evaluating immune-based therapeutic approaches in myeloid neoplasms. The immune checkpoint molecule TIM-3 (encoded by the gene HAVCR2) is a target of interest in myeloid neoplasms given its expression on leukemic stem cells as well as several subsets of immune cells including T cells, monocytes, dendritic cells and NK cells. Sabatolimab (MBG453) is an investigational IgG4 anti-TIM-3 antibody currently under evaluation for myeloid neoplasms. In this exploratory study, we sought to characterize the effects of sabatolimab combined with the hypomethylating agent (HMA), azacitidine, on the immune landscape using single cell sequencing of samples from subjects with MDS and CMML treated with the combination from a previously reported phase 1b study (NCT03066648; Brunner et al 2022). Methods Following IRB approval, single-cell RNA sequencing (scRNA-seq) and associated proteomic cellular indexing of transcriptomes and epitopes sequencing (CITE-seq) was performed on both blood (BLD) and bone-marrow (BM) derived samples from subjects with: 1) MDS (n=3) or CMML (n=2) treated with HMA therapy alone as part of standard care; 2) ND MDS (n=5) or CMML (n=3) treated with azacitidine combined with sabatolimab; and 3) relapsed/refractory MDS (n=3) treated with sabatolimab alone. Paired BLD and BM samples as well as samples from serial treatment time points were selected whenever feasible (Fig 1). A total of 206,183 cells from BM and 172,421 cells from BLD post-quality control filtering were analyzed. Further, scRNAseq data from normal BLD(200,000 cells) and BM(240,650 cells) generated as part of the human cell atlas (https://data.humancellatlas.org/explore/projects/cc95ff89-2e68-4a08-a234-480eca21ce79) were integrated with this dataset to assist with immune cell subset characterization, enable downstream comparisons to healthy hematopoietic cells, and to help define cell subsets associated with disease states. Results In baseline samples from subjects with MDS treated with azacitidine-sabatolimab, we found that an increased baseline abundance of interferon-responsive CD8 T cells in both the BM and BLD (FDR<0.1) was associated with response to therapy. An increased baseline abundance of plasmacytoid dendritic cells and a granulocyte population also suggested an association with response to therapy. Differential gene expression analysis of baseline samples from responding versus non-responding subjects with MDS treated with azacitidine-sabatolimab showed a higher baseline expression of TNF and IFNG in BM CD8 T cells and up-regulated expression of MHC-II machinery ( HLA-DRB1, HLA-DPA1, CD74) in myeloid cell subsets in responding subjects (FDR <0.1), suggesting that pre-treatment ability of these cells to be involved in antigen-presentation may play a role in response. In subjects with CMML treated with azacitidine-sabatolimab, we observed dynamic changes in cellular abundances and gene expression when comparing baseline to post-therapy timepoints. Specifically, BM CD8 T cell subsets showed an up-regulation of cytotoxicity genes ( GZMA), interferon response genes ( IFIT2, IFITM1, IFITM2) and IL32 cytokine post-therapy (FDR <0.1). When evaluating myelomonocytic cell populations, we observed an increase in CD16 monocytes post-treatment. Additionally, dendritic cells and monocytes showed an up-regulation of interferon response genes ( IFIT1, OAS1, IFI27). Conversely, we observed a down-regulation of metallothionein genes ( MT1E, MT1G, MT2A) as well as a collection of transcription factors ( NR4A1, FOSL2, JUN, CEBPB, CEBPD), NF-κB inhibitors ( NFKBIZ, NFKBIA) and CXCL8 in the post-treatment samples (FDR <0.1). Conclusions Our study provides one of the most comprehensive evaluations of the cellular dynamics of anti-TIM3 immunotherapy in patients to date, allowing for the nomination of novel putative predictive biomarkers of response and identification of potential immunomodulatory mechanisms induced by the combination of sabatolimab with azacitidine in MDS and CMML for further future analysis.

Targeting SLC3A2 Sensitizes AML Cells Towards NK Cell-Mediated Killing

Acute myeloid leukemia (AML) is an aggressive hematological malignancy with poor prognosis; hence, new therapeutic strategies are urgently needed. Natural Killer (NK) cells play a key role in tumor immune surveillance, but their anti-tumor activity in AML is often attenuated due to immunosuppressive effects of the malignant cells. Thus, strategies to restore NK cell function has therapeutic potential by boosting endogenous NK cells. In particular, the identification of cell surface proteins on AML cells that inhibit NK cell-mediated killing may reveal new targets for antibody-based therapies. To identify such targets, we performed a pooled CRISPR screen directed to 1389 cell surface genes in Mono-mac-6 (MM6), a human AML cell line. The MM6 cells were co-cultured with primary human NK cells for four days in media supplemented with the cytokines IL-2 and IL-15. Among the top hits were several genes coding for MHC class I molecules, which are known negative regulators of NK cells, demonstrating that the screen was robust. Notably, the screen also identified that SLC3A2 disruption sensitized the MM6 cells towards NK cell-mediated killing. SLC3A2 encodes the heavy chain of the transmembrane protein CD98 (CD98hc), which plays a key role in integrin signaling, regulation of intracellular calcium and the transport of L-type amino acids. CRISPR-mediated deletion of SLC3A2 in MM6 cells resulted in a two-fold downregulation of the transcripts of several inhibitory ligands of NK cells, including HLA-A, HLA-B, HLA-C and HLA-E. Upon co-culture, loss of SLC3A2 expression in MM6 cells induced an upregulation of the degranulation marker CD107a on NK cells (p<0.01), resulting in increased killing of the AML cells. CD98hc consists of two functional domains - one responsible for integrin signaling and the other responsible for amino acid transport. To identify which of these processes affect the sensitivity of AML cells towards NK cell-mediated killing, we performed rescue experiments by overexpression of mutated SLC3A2 cDNAs following SLC3A2 knockdown in MM6 cells. Overexpression of SLC3A2 wild-type or an integrin signaling deficient SLC3A2 cDNA rescued the inhibitory effect on NK cells. In contrast, the SLC3A2 variant that lacked the amino acid transportation domain failed to inhibit NK cells. These findings suggest that it is the amino acid transportation function of SLC3A2 that regulates the sensitivity of AML cells to NK cells. To validate these findings, we cultured MM6 cells in media deprived of three key amino acids (leucine, isoleucine and phenylalanine) transported across the plasma membrane by CD98. Consistent with our previous findings, culturing the MM6 cells in the amino acid deprived media resulted in a two-fold downregulation of HLA class I molecules (p<0.001) accompanied by an increased killing by NK cells. To assess the clinical relevance of these findings, we measured CD98hc expression on AML patient samples and corresponding normal bone marrow cells. CD98 levels were about 1.8-fold higher (p<0,001) in the AML samples (n = 30) compared to normal bone marrow cells (n = 5). Treating the AML patient cells with a monoclonal antibody targeting CD98 resulted in a 1.63-fold increase (p<0.0001) in NK cell-mediated killing. Corresponding treatments using normal bone marrow cells resulted in a 1.36-fold (p<0.01) increase in NK cell-mediated killing. Intriguingly, the CD98 antibody also negatively affected the viability (1.41-fold, p<0.0001) of AML patient cells in the absence of NK cells, this effect was not observed on normal bone marrow cells. Taken together, we here performed CRISPR screening on AML cells co-cultured with NK cells and identified SLC3A2 as a novel regulator of NK cells. Mechanistically, it is the amino acid transport function of SLC3A2 that regulate the sensitization of AML cells towards NK cell killing. Targeting of CD98 using a monoclonal antibody selectively increased NK cell-mediated killing of AML patient cells compared to normal bone marrow cells. These findings highlight CD98 as a new promising target on AML that boost NK cell-mediated tumor immune surveillance.

Novel Hypomethylating Agent Ntx-301 Exhibits Anti-Leukemia Activity in Venetoclax-Resistant and TP53-Mutant AML

The FDA approved Bcl-2 inhibitor venetoclax/hypomethylation agent (HMA) combination has greatly improved outcomes in patients with AML. However, despite of high response rates, the median overall survival is only 14.7 months and 2.5 months post relapse. Novel therapies in therapy-resistant AML are urgently needed. Multiple mechanisms contribute to resistance to venetoclax therapies including mutations of oncogenic kinases, leading to constitutive activation of the FLT3, SHP2, and RAS signaling cascade and upregulation of antiapoptotic Bcl-2 proteins, and mutations of TP53. We here investigate the therapeutic efficacy of NTX-301 (5-aza-thio 2'-deoxycytidine) (PinotBio, Inc.), a novel HMA in venetoclax-resistant and TP53-mutant AML. NTX-301 greatly reduced the levels of DNMT1 and decreased viability in various AML cells, markedly more effective than 5-azacytide (5-Aza). NTX-301 decreased Mcl-1 and effectively induced cell death in AML cells overexpressing Mcl-1 and Bcl-2A1, both of which are upregulated by various kinases and resistant factors to venetoclax; in AML cells with intrinsic or acquired resistance to venetoclax; and in AML cells and stem/progenitor cells obtained from patients who were resistant/relapsed from venetoclax-based therapies. Furthermore, cell death induction in aforementioned cells was synergistically enhanced when NTX-301 was combined with venetoclax. NTX-301 alone or venetoclax/NTX-301 combination had minimal activity in normal bone marrow cells and bone marrow CD34 + stem/progenitor cells. Importantly, using a PDX model developed from an AML patient who relapsed from venetoclax/HMA, NTX-301 alone demonstrated great anti-leukemia activity and significantly extended survival (167 vs 114 days of control, P=0.0001), while venetoclax or venetoclax/5-Aza did not. CyTOF analysis of mouse bone marrow cells collected at the end of 5 week treatment showed that NTX-301 and even more so NTX-301/venetoclax effectively decreased CD45 + leukemia blast cells and CD34 +CD38 +/CD34 +CD38 - AML stem/progenitor cells, while venetoclax or venetoclax/5-Aza had not effects. We observed that NTX-301 activated p53 in TP53 wild-type AML cells. We also observed that NTX-301 increased p73 and decreased MDM2 in TP53-mutant cells and showed activities in TP53-mutant leukemia cells. NTX-301 and venetoclax combination synergistically induced cell death and decreased viable cells in AML cells and stem/progenitor cells with TP53 mutations. Additionally, NTX-301 treatment increased caspase-8 and cleaved-caspase-8 in AML cells independent of TP53 mutation status, consistence with reports showing caspase-8 hypermethylation in AML and supporting that activation of caspase-8-mediated extrinsic apoptosis as a mechanism of NTX-301 action. In conclusion, our data suggest that NTX-301 has more potent anti-leukemia activities compared to current HMA in clinic and synergizes with venetoclax in venetoclax-resistance and TP53-mutant AML and AML stem/progenitor cells, and warrants clinical evaluation