Marrow Cells Research Articles

Leukolectins support lifelong innate immunity in lower vertebrates, and reveal dichotomies of several leukocytic lineages

Innate immunity is vital for animal homeostasis and survival. First-line immuno-defense for fish larvae involves mucus enriched with leukolectin (LL) secreted by dermal lectocytes. Later during the critical transition from yolk-nutrition to feeding, additional larval immuno-protection in zebrafish (zF) is provided by macrophages containing LL (lectophages). This work investigated new LL-expression in embryos and in blood, structures of fish leukocytic LL and LL-genes, and LL-presence in chicken leukocytes. In zF-embryos, lectophages appear ∼10 hpf, while later, cells co-expressing myeloperoxidase- and LL-mRNA were detected (∼19 hpf). Furthermore, protein-extracts of Atlantic salmon (Ssal) leukocytes contained LL-proteins, compartmentalized in the cytosol. Cloning and sequencing revealed 94 % nt-sequence identity between variants of Ssal-leukolectins. Highly conserved LLs allowed production of epitope-specific anti-LL IgGs. Immuno-fluorescence-analysis demonstrated that most Ssal-bloodcells were LL-negative, but both some large cells with protrusions and some small, rounded cells did express LL. Immunoperoxidase-staining method confirmed LL-expression in some Ssal-leukocytes, identified as macrophages, PMN-leukocytes, thrombocytes and dendritic cells. However, closer examination revealed a dichotomy of these cell-categories into either LL-positive, or LL-negative variants. In situ hybridization demonstrated profuse LL-expression in Ssal head kidney interstitial tissue, while LL-transcripts were absent in large kidney tubules. Both hematopoietic (non-pigmented) marrow cells and melano-macrophages expressed LL-mRNA, implying that leukolectins provide lifelong innate immuno-protection. PCR-amplification using Ssal-leukocytic DNA as template, and direct sequencing yielded a leukocytic ll-gene. Some cells in salmon, cod, halibut, oikopleura and zebrafish embryos express LL-proteins and/or LL-mRNA, and LL-mRNA is detected in salmon, cod and chicken leukocytes. However, current genomes for these species lack recognizable LL-loci except the Ssal_v3.1 Genome-assembly. The data demonstrate an unexpected dichotomy of some leukocyte lineages into LL-positive or LL-negative cell-variants. Such dichotomies suggest exploring differential impacts from the duplicated leukocyte-lineages in health and disease.

Analysis and Interpretation of Automated Blood Count in the Treatment of Chronic Paracoccidioidomycosis.

Blood count is crucial for assessing bone marrow's cell production and differentiation during infections, gaging disease severity, and monitoring therapeutic responses. The profile of blood count in chronic forms of paracoccidioidomycosis (PCM) has been insufficiently explored. To better understand the changes in hematological cells in different stages of the PCM chronic form, we evaluated the blood count, including immature blood cells in automated equipment, before and during the treatment follow-up of 62 chronic PCM patients. Predominantly male (96.8%) with an average age of 54.3 (standard deviation SD 6.9) years, participants exhibited pre-treatment conditions such as anemia (45.2%), monocytosis (38.7%), and leukocytosis (17.7%), which became less frequent after clinical cure. Anemia was more prevalent in severe cases. Notably, hemoglobin and reticulocyte hemoglobin content increased, while leukocytes, monocytes, neutrophils, immature granulocytes, and platelets decreased. Chronic PCM induced manageable hematological abnormalities, mainly in the red blood series. Monocytosis, indicating monocytes' role in PCM's immune response, was frequent. Post-treatment, especially after achieving clinical cure, significant improvements were observed in various hematological indices, including immature granulocytes and reticulocyte hemoglobin content, underscoring the impact of infection on these parameters.

A Multi-Color Flow Cytometric Assay for Quantifying Dinutuximab Binding to Neuroblastoma Cells in Tumor, Bone Marrow, and Blood.

GD2, a disialoganglioside, is present on the surface of most neuroblastomas, as well as on some other cancers, such as melanoma and osteogenic sarcoma. The anti-GD2 antibody ch14.18 (dinutuximab) has an FDA-registered indication for use as maintenance therapy for high-risk neuroblastoma with cytokines and 13-cis-retinoic acid after myeloablative therapy. Recent studies using immunohistochemistry of tumor or tumor cells in marrow have shown that some neuroblastomas are negative for GD2. Dinutuximab and other anti-GD2 antibodies are increasingly used in combination with cytotoxic chemotherapy for treating relapsed neuroblastoma, so it is important to be able to identify patients with tumor cells with low GD2 expression, as such patients may experience toxicity but not benefit from the antibody therapy. As the most common clinical samples available for relapsed neuroblastoma are bone marrow aspirates, we developed a method to quantify dinutuximab binding density and the frequency of neuroblastoma cells positive for the antibody in bone marrow aspirates. Here, we describe a multi-color flow cytometry assay that employs non-GD2 antibodies to identify neuroblastoma cells in a mixed population (tumor, bone marrow, or blood) and an anti-GD2 antibody to quantify both the frequency and density of GD2 expression on neuroblastoma cells.

Novel covalent CDK7 inhibitor potently induces apoptosis in acute myeloid leukemia and synergizes with Venetoclax

IntroductionThe emergence of resistance to the highly successful BCL2-directed therapy is a major unmet need in acute myeloid leukemia (AML), an aggressive malignancy with poor survival rates. Towards identifying therapeutic options for AML patients who progress on BCL2-directed therapy, we studied a clinical-stage CDK7 inhibitor XL102, which is being evaluated in solid tumors (NCT04726332).Materials and methodsTo determine the anti-proliferative effects of XL102, we performed experiments including time-resolved fluorescence resonance energy transfer, target occupancy, cell cycle and apoptosis-based assays. We also included genetically characterized primary myeloid blasts from de novo and relapsed/refractory AML patients. For mechanistic studies, CRISPR/Cas9 mediated knockout of CDK7 and c-Myc and immunoblotting were performed. NOD/SCID orthotropic and subcutaneous AML xenografts were used to determine anti-leukemic effects. To assess the synergistic effects of XL102 with Venetoclax, we performed RNA sequencing and gene set enrichment analysis using Venetoclax sensitive and resistant model systems.ResultsXL102, a highly specific, orally bioavailable covalent inhibitor of CDK7. Inhibitory effect on CDK7 by XL102 in primary myeloid blasts (n = 54) was in nanomolar range (mean = 300 nM; range = 4.0-952 nM). XL102 treated AML cells showed a reduction in phosphorylation levels of Serine 2/5/7 at carboxy-terminal domain of RNA polymerase II. T-loop phosphorylation of CDK1(Thr161) and CDK2(Thr160) was inhibited by XL102 in dose-dependent manner leading to cell-cycle arrest. c-Myc downregulation and enhanced levels of p53 and p21 in XL102 treated cells were observed. Increased levels of p21 and activation of p53 by XL102 were mimicked by genetic ablation of CDK7, which supports that the observed effects of XL102 are due to CDK7 inhibition. XL102 treated AML xenografts showed remarkable reduction in hCD45 + marrow cells (mean = 0.60%; range = 0.04%-3.53%) compared to vehicle control (mean = 38.2%; range = 10.1%-78%), with corresponding increase in p53, p21 and decrease in c-Myc levels. The data suggests XL102 induces apoptosis in AML cells via CDK7/c-Myc/p53 axis. RNA-sequencing from paired Venetoclax-sensitive and Venetoclax-resistant cells treated with XL102 showed downregulation of genes involved in proliferation and apoptosis.ConclusionTaken together, XL102 with Venetoclax led to synergistic effects in overcoming resistance and provided a strong rationale for clinical evaluation of XL102 as a single agent and in combination with Venetoclax.

Adults Myelodysplastic Syndromes in Algeria: A Study by the Algerian MDS Group

Introduction: Myelodysplastic syndromes (MDS) are a group of haematological disorders, whose diagnosis is based mainly on cytological studies of blood and marrow cells and cytogenetic analysis. Moreover, national epidemiological data on MDS are very scarce, especially in Maghreb countries where the population is on average younger than in Europe or the USA. The objective of the present study was to describe demographic and clinical features and the overall survival of patients with MDS in Algeria. Materials and Methods: This study is retrospective and national multicenter (n=19 centres), performed between 2014 to 2019. The evaluation was performed using EPI-INFO and SPSS version 21 software. Survival data were calculated using the Kaplan-Meier method and comparison of survival curves using the Log Rank test. Univariate and multivariate analysis of survival was performed using the Cox regression method. The study has been approved by the Ethical and Scientific Council of the participating hospitals. The closing date of the study is 31/12/2019. Results: A total of 670 patients with newly diagnosed MDS have been identified. The average number of new cases was 112/year, with an annual progression rate of 19%. Demographics show a slight female predominance (M/F of 317/353=0, 89; sex ratio F/M=1.11). The median follow-up was 29,3 months (range, 1 to 77 months). The overall median age was 69 years (range 16-96). The crude mean annual incidence rate was 0, 38 per 100,000 inhabitants aged ≥15 years old and it was 0, 17/100,000 in men and 0, 21/100,000 in women. Overall survival was 39 months. According to the IPSS score, the high-risk forms are low and their overall survival was 15 months. The rate of transformation into acute myeloid leukaemia (AML) is 32%. Conclusion: This national epidemiological survey shows an annual progression rate of 19% and an increase in incidence from 0.007/100.000 in 2005 to 0.45/100.000 in 2019.

Secondary plasma cell leukemia: Case report

Plasma cell leukemia (PCL) is a rare lymphoproliferative disorder characterized by monoclonal proliferation of plasma cells in marrow and peripheral blood. It is defined by a blood plasma count greater than 2 G/l or a plasma cell count greater than 20% of white blood cells. There are two forms of PCL: primary PCL (primary plasma cell leukemia or PCL-P), which occurs de novo and is diagnosed by blood dissemination, and secondary PCL (secondary plasma cell leukemia or SPL), which is the result of the evolution of multiple myeloma (MM). We report an observation of secondary plasma cell leukemia in a 42-year-old female patient diagnosed at the hematology laboratory of the Mohamed VI University Hospital in Marrakech. The patient was being followed up for chronic end-stage renal failure in whom the etiological assessment of a hypercalcemia of fortuitous discovery revealed MM for which she was put on chemotherapy according to the Velcade-Thalidomide-Dexamethasone protocol with a good evolution after 6 courses. Two and a half years later, the patient presented with anemia and spinal pain. The biological work-up showed a normocytic normochromic anemia at 5.4 g/dl and hypercalcemia at 150 mg/l. The blood smear showed the presence of 22% of circulating plasma cells, the majority of which were dystrophic, in favor of secondary plasma cell leukemia. Patients diagnosed with multiple myeloma who have undergone treatment and have a stable disease should have regular surveillance, hence the interest of the blood smear examination in all multiple myeloma.

Study Genotoxic and Cytotoxic effect of the fungicide Propamocarb- HCl in Albino mice

The study was designed to evaluate genotoxicity and cytotoxicity of the fungicide probamocarb- HCl using Swiss white mice Mus musculus .
 The evaluation of treatment effects with the dosage 1355 and 2349 mg/kg .bw of the fungicide was depended on genomarkers (Micronuclei in bone- marrow cells, evaluation of DNA damage in liver and bone-marrow cells using comet assay).
 The results showed significant linear increase at p≤0.05 between time of treatment and micronuclei which reached 420.40 ± 12.21 with replicate treatment of the dosage 1355 mg/kg.bw of the fungicide compared with 4.80 ± 2.10 for negative control and 592.60±5.83 with single dosage of 2493 mg/kg. bw of the fungicide. Change in the percentage of polychromatic erythrocytes also observed which have increased in linear manner with the increased time of treatment. It reached 11.23% with replicate treatment of the dosage 1355 mg/kg.bw of the fungicide and 10.62% with the single treatment of the dosage 2493 mg/kg.bw. compared with 9.71 for negative control. The number of micronucleated polychromatic erythrocytes was significantly increased in linear manner with the replicate treatment of the dosage 1355 mg/kg.bw of the fungicide which reached 310.60 ± 1.36 and 243.60 ± 5.29 with the single dosage of 2493 mg/kg.bw of the fungicide compared with 2.80 ± 1.06 for negative control.
 Treatment with Propamocarb-HCl induced damage in DNA of liver and bone-marrow cells. The value of OTM reached 58.40 ± 2.01 in bone-marrow cells with the single dosage of 2493 mg/kg.bw compared with 3.20 ± 0.66 for negative control. While it reached 61.80 ± 7.57 compared with 8.00 ± 1.38 for negative control in liver cells. The percentage of cells with damaged DNA reached 25.88% in bone-marrow and 30.62% in liver.

Clonal Hematopoiesis Evolves into Dendritic Cell Neoplasm through the Acquisition of UV Mutations in the Skin

Background Expansion of blood cells derived from a single mutated blood stem cell - clonal hematopoiesis - often precedes leukemic transformation. However, the circumstances that promote or prevent transformation are poorly understood. Here, we investigate the evolution of clonal hematopoiesis into blastic plasmacytoid dendritic cell neoplasm (BPDCN), an aggressive blood cancer that often presents with malignant cells isolated to the skin (referred to as skin-only disease). We gain a better understanding of the influence of site-specific selective pressures and the role of TET2 mutations, which may inform interventions to prevent disease progression. Methods We analyzed bone marrow and skin from 13 BPDCN patients using targeted, exome, and whole-genome sequencing, with germline controls. We performed multimodal single-cell sequencing (10x 3' v3.1 single-cell RNA-seq with genotyping of expressed variants) on bone marrow from 6 healthy donors (20,411 cells), 5 skin-only BPDCN patients with malignant cells in the skin but not conventionally detectable in marrow (36,018 cells), and 6 BPDCN patients with overt bone marrow involvement (30,582 cells). For functional studies, we targeted Tet2 using CRISPR/Cas9 in mouse blood progenitor cells immortalized by an estrogen receptor hormone binding domain fused to HoxB8, which undergo synchronized differentiation to myeloid and dendritic cells upon estrogen withdrawal. We used CellRanger, Seurat, RandomForest machine learning, and the R tidyverse package collection for bioinformatics. Results Genetics allowed us to reconstruct the evolution of BPDCN across bone marrow and skin (representative patient, panel a). Invariably, we detected founder mutations in marrow, including TET2 inactivation in 9/13 patients (two mutations, likely bi-allelic, in 5/9). Single-cell sequencing of marrow from 5 skin-only patients showed that pre-malignant mutant stem cells contributed to multiple lineages, and that TET2 mutated clones were myeloerythroid-biased. We generated a gene expression signature to identify rare malignant BPDCN cells in marrow of skin-only patients. In 3/5 patients, we identified rare cells (0.03-0.19%) with the transcriptional profile of fully transformed cells, and concurrent single-cell genetic analysis suggested that these cells originated from the skin tumor (panel b). To assess whether disseminated skin tumor cells establish bone marrow disease, we quantified mutational signatures: skin tumors and malignant BPDCN marrow cells in 5/5 patients harbored UV-induced mutations, likely acquired in a single skin-resident plasmacytoid dendritic cell (pDC) during malignant transformation. Finally, we edited Tet2 in mouse myelo-dendritic progenitors and tested the response to UV irradiation in vitro. In addition to observing that Tet2 inactivation increases dendritic cell commitment, the more striking result was a survival advantage of Tet2-mutated cells in the presence of UV (n = 6, fold change 1.803, P = 0.0012). These results highlight an unexpected function of TET2 that may predispose mutated pDCs to survive UV-induced DNA damage in the skin, leading to disease progression. Conclusion The complex evolution of BPDCN traverses multiple anatomic sites prior to full transformation. This allows for accurate sequencing of oncogenic events. Disease evolution frequently starts with acquisition of a leukemia-associated mutation in a multipotent blood stem cell, leading to clonal expansion. In the most frequently observed scenario, bi-allelic TET2 inactivation promotes myeloerythroid bias, increases dendritic cell commitment, and confers resistance to UV-mediated cell death. Subsequent evolution of skin-resident mutant pDCs leads to a high burden of UV-induced mutations and ultimate transformation. Dissemination of malignant cells, which can be detected by integrating single-cell gene expression and mutation profiles, initiates "retrograde" bone marrow disease. Our discovery that multiple anatomic sites - and local selective pressures - play a role in disease progression provides a template to study multi-site evolution of clonal hematopoiesis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Over expression of mTOR gene predicts overall survival in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) is defined as heterogenous disease, it contains heterogenous leukemic stem cells with various degree of cell differentiation. The perturbation of genes involved in myeloid progenitor cell growth, differentiation and proliferation lead to morphologic dysplasia, maturation arrest, ineffective hematopoiesis hence the cytopenias and propensity to develop into acute myeloid leukemia (AML). Heterogeneous subsets of MDS patients have been defined by their clinical and biologic abnormalities. These different features lead to the development of different prognostic system; however, these approaches are limited in predicting clinical course, and management of patients remains challenging given the uncertainty of the time course of disease progression. It is of importance to identify transcriptomic marker causing maturational and differentiation arrest which could help in understanding the pathogenesis of disease. We have studied differential gene expression profiles (GEPs) in CD34 + marrow cells from myelodysplastic syndrome (MDS) patients (n = 14) and control CD34 + cells using Affymetrix Human Clariom S microarray with 20,000 well annotated genes. We found 4165 genes significantly (p < 0.05) differentially expressed in MDS. Using stringent bioinformatics analyses, we were able to identify few genes (MAPK8, JUNB, mTOR) which were differentially upregulated i.e. 5.39, 73.61 and 2.7 fold change observed in MDS than control and also validated (n = 60) these genes by RT - qPCR. Kaplan - Meier survival analysis indicated that MAPK8 and JUNB could be poor prognostic marker as patients with increased expression showed poor survival, whereas surprisingly mTOR increased expression proved to be good prognostic marker. The correlation analysis showed that the level of gene (MAPK8, JUNB, mTOR) expression was significantly (p ≤ 0.05) associated with frequency of genetic lesions. Interestingly the increased expression of MAPK8 was significantly accompanied with ASXL1 gene mutation. Our study showed an elevation of TNF and AMPK signalling pathways in MDS. TNF signalling might be mediating the proliferative advantage to myeloid clonal cells (mutation carrying cells) over normal cells, whereas, AMPK signalling could be acting as protector against it (favouring normal cells). Hence it would be interesting to explore the functions and pathways associated with mTOR, AMPK, MAPK8 and JUNB in myelopoiesis related diseases like MDS.

Effect of Advanced Oxidation Protein Products (AOPPs) and aging on the osteoclast differentiation of Myeloid-Derived Suppressor Cells (MDSCs) and its preliminary mechanism

To verify the osteoclast differentiation ability of MDSCs from mice of different ages and explore the effect of AOPPs on the osteoclast differentiation of bone marrow MDSCs.Bone marrow cells from C57BL/6 (a.k.a C57) mice of different ages were subjected to flow cytometry, and CD11b+Ly6C+Ly6G+ MDSCs were sorted out. After induction of osteoclast differentiation, these cells were subjected to tartrate-resistant acid phosphatase (TRAP) and F-actin. MDSCs from bone marrows of old mice were injected into the tibial medullary cavity of young mice. One week later, the bone marrows were subjected to histological examination, TRAP, and cell count. MDSCs from bone marrows of old mice were sorted for induction of osteoclast differentiation, intervened with reactive oxygen species (ROS) scavenger, inducible nitric oxide synthase (iNOS) inhibitor, and nitric oxide (NO) scavenger, and then subjected to TRAP. 8-weeks-old C57 mice were injected with the same concentrations of either AOPPs or mouse serum albumin (MSA). Four weeks later, MDSCs from bone marrows were sorted and subjected to induction of osteoclast differentiation, followed by IHC staining and TRAP. MDSCs of 8-weeks-old C57 mice were extracted and subjected to in vitro induction of osteoclast differentiation with different concentrations of AOPPs, followed by TRAP training.The number of MDSCs in the bone marrows of old mice was significantly higher than that in young mice. MDSCs from bone marrows of old mice differentiated into large multinucleated TRAP+ osteoclasts, which were significantly different from those in the middle-aged and young mice in terms of cell quantity and morphology. The actin rings formed in the differentiated osteoclasts from MDSCs of bone marrows were densely distributed in the whole field of view, which were significantly denser than those in the middle-aged and young mice. After injection of MDSCs of old mice, the number of TRAP + osteoclasts in the tibial medullary cavity of young mice was significantly increased. NO inhibitor can significantly inhibit the osteoclast differentiation capacity of MDSCs from bone marrows of old mice. In vivo treatment with AOPPs significantly increased the proportion of MDSCs in the bone marrow, which is up to 55.2%. After injection of AOPPs in 8-week-old mice and induction of osteoclast differentiation from the MDSCs, the ratios of CD11b+ and Gr1+ cells were significantly higher than that in the control and MSA groups but was not significantly different from that in the 15-month-old mice. Upon in vitro treatment with different concentrations of AOPPs, the MDSCs did not show any sign of osteoclast differentiation.MDSCs can directly undergo osteoclast differentiation, the capacity of which is stronger in MDSCs of bone marrows of old mice; the NO pathway is a potential mechanism underlying this phenomenon. In vivo but not in vitro AOPPs treatment can induce osteoclast differentiation of MDSCs, indicating there might be other factors in the body that can interact with AOPPs to induce osteoclast differentiation of MDSCs.

IGH cytogenetic abnormalities can be detected in multiple myeloma by imaging flow cytometry

AimsCytogenetic abnormalities involving the IGH gene are seen in up to 55% of patients with multiple myeloma. Current testing is performed manually by fluorescence in situ hybridisation (FISH) on purified...

Role of C-reactive protein in the bone marrow of Modic type 1 changes.

Modic type 1 changes (MC1) are vertebral bone marrow lesions and associate with low back pain. Increased serum C-reactive protein (CRP) has inconsistently been associated with MC1. We aimed to provide evidence for the role of CRP in the tissue pathophysiology of MC1 bone marrow. From 13 MC1 patients undergoing spinal fusion at MC1 levels, vertebral bone marrow aspirates from MC1 and intrapatient control bone marrow were taken. Bone marrow CRP, interleukin (IL)-1, and IL-6 were measured with enzyme-linked immunosorbent assays; lactate dehydrogenase (LDH) was measured with a colorimetric assay. CRP, IL-1, and IL-6 were compared between MC1 and control bone marrow. Bone marrow CRP was correlated with blood CRP and with bone marrow IL-1, IL-6, and LDH. CRP expression by marrow cells was measured with apolymerase chain reaction. Increased CRP in MC1 bone marrow (mean difference: +0.22 mg CRP/g, 95% confidence interval[CI] [-0.04, 0.47], p = 0.088) correlated with blood CRP (r = 0.69, p = 0.018), with bone marrow IL-1β (ρ = 0.52, p = 0.029) and IL-6 (ρ = 0.51, p = 0.031). Marrow cells did not express CRP. Increased LDH in MC1 bone marrow (143.1%, 95% CI [110.7%, 175.4%], p = 0.014) indicated necrosis. A blood CRP threshold of 3.2 mg/L detected with 100% accuracy increased CRP in MC1 bone marrow. In conclusion, the association of CRP with inflammatory and necrotic changes in MC1 bone marrow provides evidence for a pathophysiological role of CRP in MC1 bone marrow.

Thymic plasmacytoma presenting as polyneuropathy and revealing multiple myeloma: a case report

BackgroundMultiple myeloma (MM) is the most frequent malignant plasma cell disorder with proliferation of neoplastic plasma cells in the bone marrow or other tissue, most commonly in the upper aerodigestive tract. The invasion of the thymus is exceptional. Neurological complications are usual, but represent exceptionally the revealing symptom.Case presentationWe report a case of polyneuropathy revealing a thymic plasmacytoma as a mediastinal invasion of MM in a 48-year-old woman. She was admitted after developing progressive ascending distal paresthesias and weakness in lower limbs. Examination showed symmetrical distal sensorimotor impairment with axillary and inguinal adenopathies. Electroneuromyography revealed a sensorimotor length-dependent neuropathy. Serum protein electrophoresis showed monoclonal protein peak in β-γ globulin region. Immunoelectrophoresis showed IgA lambda monoclonal gammapathy. Myelogram and bone marrow biopsy revealed plasmocytosis of 5%. Chest computed tomography showed a histologically confirmed thymic plasmacytoma associated with a lytic lesion of the 5th rib leading to the diagnosis of MM.ConclusionsThe association between a thymic plasmacytoma and peripheral neuropathy is rare and a workup for MM is necessary to guide therapeutic management.

Effective tumor cell abrogation via Venetoclax-mediated BCL-2 inhibition in KMT2A-rearranged acute B-lymphoblastic leukemia

Dysregulation of the intrinsic BCL-2 pathway-mediated apoptosis cascade is a common feature of hematological malignancies including acute B-lymphoblastic leukemia (B-ALL). The KMT2A-rearranged high-risk cytogenetic subtype is characterized by high expression of antiapoptotic protein BCL-2, likely due to the direct activating binding of KMT2A fusion proteins to the BCL2 gene. The BCL-2 inhibitor venetoclax (VEN) has proven great clinical value in other blood cancers, however, data on B-ALL is sparse and past studies have not so far described the effects of VEN on gene and protein expression profiles. Using cell lines and patient-derived in vivo xenograft models, we show BCL-2 pathway-mediated apoptosis induction and decelerated tumor cell counts in KMT2A-rearranged B-ALL but not in other cytogenetic subtypes. VEN treatment of cell line- and patient-derived xenografts reduced blast frequencies in blood, bone marrow, and spleen, and tumor cell doubling times were increased. Growth rates are further correlated with VEN concentrations in blood. In vitro incubation with VEN resulted in BCL-2 dephosphorylation and targeted panel RNA sequencing revealed reduced gene expression of antiapoptotic pathway members BCL2, MCL1, and BCL2L1 (BCL-XL). Reinforced translocation of BAX proteins towards mitochondria induced caspase activation and cell death commitment. Prolonged VEN application led to upregulation of antiapoptotic proteins BCL-2, MCL-1, and BCL-XL. Interestingly, the extrinsic apoptosis pathway was strongly modulated in SEM cells in response to VEN. Gene expression of members of the tumor necrosis factor signaling cascade was increased, resulting in canonical NF-kB signaling. This possibly suggests a previously undescribed mechanism of BCL-2-independent and NF-kB-mediated upregulation of MCL-1 and BCL-XL. In summary, we herein prove that VEN is a potent option to suppress tumor cells in KMT2A-rearranged B-ALL in vitro and in vivo. Possible evasion mechanisms, however, must be considered in subsequent studies.

Abstract 5780: Functions of genetic variation on gene expression and survival in multiple myeloma

Abstract Multiple myeloma (MM) is a malignancy of plasma cells in the bone marrow and the second most common hematological malignancy. MM is characterized by a high degree of heterogeneity and has a hereditary genetic component with relatives of MM patients having a higher risk of disease development. Additionally, MM displays a disparity in occurrence and mortality among sexes with males having a higher risk than females. Although new therapies have significantly improved survival rates, MM remains incurable as most patients experience relapses. Therefore, understanding the genetic control underlying complex outcomes is important for the development of better treatments for MM patients. While the majority of research has been focusing on protein-coding regions of the genome, recent studies have started to illuminate the role of non-coding variations on MM. For example, many of the 176 GWAS risk loci associated with MM susceptibility are located within or adjacent to regulatory regions, indicating a role in transcriptional regulation. However, how the non-coding genetic variations affect gene expression, tumor etiology and outcome is poorly understood. To better understand the genetic and biological underpinnings of MM, we utilize genomic approaches to examine non-coding germline and somatic effects on gene expression and cancer outcome. First, we analyzed whole-genome sequencing (WGS) from peripheral blood and WGS and RNA-seq from baseline tumor specimens of 607 participants from the CoMMpass longitudinal study of the Multiple Myeloma Research Foundation. Using both joint and sex-stratified analyses, we detected 6,504 unique germline variants associated with changes in gene expression, i.e. expression quantitative trait loci (eQTLs), corresponding to 4,598 unique eGenes. Among the identified eQTLs, 33.76% exhibit sex-specific effects and 654 variants are associated with genes that influence survival in the CoMMpass cohort. In addition to the germline eQTLs, we identified 266 somatic eQTLs, corresponding to 208 eGenes. Among the identified eQTLs, 566 germline and 88 somatic eQTLs overlap with an ATACseq peak in MM cell lines. Second, we selected a set of eQTLs to validate their functions in transcriptional activity in MM cell lines (2 females, 2 males) using CRISPR interference screens (CRISPRi) with a scRNA-seq readout. By analyzing global transcriptional changes, we identify potential downstream targets that may play essential roles in MM genesis and progression. In conclusion, using computational and functional genomics approaches, we have identified germline and somatic eQTLs that are associated with gene expression and survival in MM, thus providing insights into how these non-coding variations contribute to tumor etiology and outcome. Citation Format: Linh T. Bui, Heini M. Natri, Lance M. Peter, Bianca Argente, Austin J. Gutierrez, Arnold Federico, Mei-I Chung, Jonathan Keats, Nicholas E. Banovich. Functions of genetic variation on gene expression and survival in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5780.

Abstract 4216: Tipifarnib inhibits the secretion of tumor-derived small extracellular vesicles and enhances the immunotherapeutic efficacy of dinutuximab in neuroblastoma

Abstract Anti-GD2 monoclonal antibody (Dinutuximab) immunotherapy has significantly improved the overall survival rate for high-risk neuroblastoma patients. However, 40% of patients fail to respond or develop resistance to the treatment, and the molecular mechanisms by which this occurs remain poorly understood. Here we utilize the syngeneic 9464D-GD2 mouse model to investigate the role of neuroblastoma tumor-derived small extracellular vesicles (sEVs) in developing resistance to the anti-GD2 monoclonal antibody Dinutuximab. Neuroblastoma-derived sEVs significantly attenuated the efficacy of Dinutuximab treatment in vivo. Mechanistically, both RNA-sequencing and flow cytometry analysis of whole tumors demonstrated that neuroblastoma-derived sEVs modulate immune cells tumor infiltration upon Dinutuximab treatment to create an immunosuppressive tumor microenvironment, which contains more tumor-associated macrophages (TAMs) and fewer tumor-infiltrating NK cells, in addition to suppression of splenic NK cell maturation in vivo. Moreover, neuroblastoma-derived sEVs suppressed Dinutuximab-mediated NK cell antibody-dependent cellular cytotoxicity in vitro. Importantly, Tipifarnib, a farnesyltransferase inhibitor that inhibits sEV secretion, drastically enhanced the efficacy of Dinutuximab in vivo and reversed the immunosuppressive effects of neuroblastoma-derived sEVs. Notably, Tipifarnib modulated immature myeloid cells in the bone marrow to disfavor the formation of CD11b+Ly6ChighLy6Glow subpopulations, which are the precursors for TAMs. Taken together, these preclinical findings uncover a novel mechanism by which neuroblastoma-derived sEVs modulate the immune system to promote resistance to Dinutuximab and suggest that Tipifarnib-mediated inhibition of sEV secretion may serve as a viable treatment strategy to improve immunotherapy in high-risk patients. Citation Format: Xiaoming Liu, Carson A. Wills, Longgui Chen, Jiawen Zhang, Yuanjun Zhao, Mi Zhou, Jeffrey M. Sundstrom, Todd D. Schell, Vladimir S. Spiegelman, Megan M. Young, Hong-Gang Wang. Tipifarnib inhibits the secretion of tumor-derived small extracellular vesicles and enhances the immunotherapeutic efficacy of dinutuximab in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4216.

Abstract 5496: Efficacy and immune profiling of PI3K delta inhibitor zandelisib (ME-401) in a preclinical chronic lymphocytic leukemia (CLL) model

Abstract Phosphatidylinositol-3 kinase (PI3K) delta signaling is essential for CLL B cell survival and proliferation. Several PI3K inhibitors are approved for treatment of CLL, but PI3K inhibitor-induced alterations in T cell function have been correlated with immune-related adverse events (irAEs). Zandelisib is a novel selective PI3Kδ inhibitor in clinical development with an intermittent dosing schedule for treatment of B cell malignancies. Here, we investigated the dynamic immunomodulatory properties of zandelisib alone or in combination with BTK inhibitor ibrutinib in normal human T cells, as well as the impact of continuous dosing of zandelisib on effector and regulatory immune cells, irAEs, and survival in a preclinical CLL murine model. Normal human T cells treated ex vivo with zandelisib, ibrutinib, or both were assessed for phenotype and function. A CLL model was initiated by transferring leukemic EμTCL1 splenocytes into syngeneic wildtype recipients. CLL-bearing mice were treated with vehicle or zandelisib (50 mpk bid or 100 mpk qd) for 3 weeks (n=5 per group). Antitumor efficacy, T cell and myeloid subsets in blood, bone marrow and spleen were analyzed. Survival was also observed in a separate cohort of mice treated with zandelisib (100 mpk qd). T cell proliferation was impaired by zandelisib (250nM) and ibrutinib (100 nM) alone, with the combination further reducing T cell proliferation. Zandelisib decreased expression of suppressive markers PD-1, CTLA-4, GITR and CD39 on iTregs; however, no further decrease was observed when zandelisib was combined with ibrutinib. Zandelisib also reduced normal T cell activation and expression of immune checkpoints PD-1 and CTLA-4 on CD4+ T cells and dose dependently inhibited T cell memory differentiation ex vivo, increasing the naïve/central to effector memory ratio. Combination with ibrutinib did not further affect T cell activation, checkpoint expression or memory differentiation. In the CLL murine model, no meaningful difference was seen between bid or qd administration of zandelisib in the reduction of tumor burden (CD19+ CD5+ CLL cells). Zandelisib treatment reduced activated T cells, antigen-experienced T cells, and Tregs. Increased naïve/central to effector memory ratio was observed in treated mice, and PD-1 was downregulated on memory T cells, indicating less T cell exhaustion. Zandelisib decreased macrophages in the spleen but did not impact myeloid-derived suppressor cells. Zandelisib treatment improved survival of CLL-bearing mice significantly when compared to vehicle group (median survival of zandelisib group 140 days vs. 105 days vehicle). In conclusion, zandelisib treatment reduced Tregs, prevented terminal memory differentiation, and T-cell exhaustion—features that have been shown to permit CLL immune evasion—demonstrated antitumor efficacy, and improved overall survival in a preclinical CLL model. Citation Format: Kamira Maharaj, Melanie Mediavilla-Varela, John J. Powers, Sandra Wiley, Angimar Uriepero, Wael Gamal, Kun Jiang, Eva Sahakian, Javier Pinilla-Ibarz. Efficacy and immune profiling of PI3K delta inhibitor zandelisib (ME-401) in a preclinical chronic lymphocytic leukemia (CLL) model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5496.

Abstract 641: Single-cell RNA sequencing of rare circulating tumor cells in precursor myeloma patients reveals molecular underpinnings of tumor cell circulation

Abstract Background: Multiple Myeloma (MM) is a hematological malignancy characterized by abnormal proliferation of terminally differentiated plasma cells (PCs) in the bone marrow (BM). MM is almost always preceded by the precursor stage smoldering multiple myeloma (SMM). BM biopsies are useful to monitor disease progression, but they are invasive and not routinely collected from patients for disease monitoring during precursor stages. Profiling circulating tumor cells (CTCs) from peripheral blood (PB) could aid early detection, disease monitoring, and biomarker identification to predict patients at high risk of progression that may benefit from early therapeutic intervention. Methods: Paired PB and BM aspirates were collected from 40 SMM patients enrolled in the PCROWD study (IRB #14-174) at Dana-Farber Cancer Institute. Malignant PCs were enriched by magnetic bead-based methods and underwent 5’ single-cell RNA sequencing (scRNA-seq) and single-cell B-cell receptor sequencing (scBCR-seq) (10x Genomics). Results: We analyzed 105,246 BM PCs and 33,234 PB PCs from 15 patients. To differentiate malignant from normal PCs, we used clonal V(D)J rearrangements, assessed by concurrent scBCR-seq. A total of 86,986 BM tumor cells and 8,718 CTCs were captured. A median of 5, 26, and 47 CTCs were present per mL of blood from low, intermediate, and high-risk SMM patients as defined by the International Myeloma Working Group (IMWG) “20/2/20” criteria, suggesting sequencing-based CTC enumeration corresponds to prognosis. High levels of driver genes commonly upregulated in patients with specific translocations, including CCND1 and MAF, were detected in both BM tumor and CTC clusters in 3 patients with t(11;14) and t(14;16) confirmed by fluorescence in situ hybridization (FISH) clinical testing, and 2 additional patients with inconclusive FISH results (Wilcoxon, q &amp;lt;10-3), supporting the idea of CTC-based prognostication. Differential expression (DE) analysis revealed 8 genes that were significantly upregulated and 3 genes that were significantly downregulated in CTCs compared to BM tumor cells robustly across 15 paired samples. Gene set enrichment analysis (GSEA) revealed genes DE in CTCs are associated with TNF-α and NF-κB signaling, which are commonly induced by extrinsic factors in the bone marrow milieu, providing insight into the biology of tumor cell circulation. Conclusions: This study highlights the utility of scRNA-seq for molecular profiling of CTCs, even in asymptomatic low tumor burden disease. Additional analyses are ongoing in the expanded cohort of 40 patients with paired samples to help gain further insight into CTC heterogeneity. Overall, this study will help enable the design of new molecular liquid biopsy-based approaches to diagnosis, disease monitoring, and biological insights to improve treatment strategies for precursor myeloma patients. Citation Format: Elizabeth D. Lightbody, Danielle T. Firer, Romanos Sklavenitis-Pistofidis, Michael Agius, Ankit K. Dutta, Michelle Aranha, Jean-Baptiste Alberge, Laura Hevenor, Nang Kham Su, Cody Boehner, Erica Horowitz, Jacqueline Perry, Anna Cowan, Hadley Barr, Anna Justis, Daniel Auclair, Catherine R. Marinac, Gad Getz, Irene Ghobrial. Single-cell RNA sequencing of rare circulating tumor cells in precursor myeloma patients reveals molecular underpinnings of tumor cell circulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 641.

Abstract 5662: Targeting N-myristoylation for therapy of adult acute myeloid leukemia

Abstract Two N-myristoyltransferases (NMTs) NMT1 and NMT2 catalyze the reaction. NMT1 is ubiquitous expressed and is essential for cell survival while NMT2 is more variably expressed and non-essential suggesting that their substrate specificity and activity levels differ. Historically, inhibition of myristoylation was suggested as a therapeutic anti-cancer target since NMTs expression were shown to be increased in numerous types of cancers and myristoylation was shown to be essential for proper localization and activity of some important proto-oncogenes such as Src Family Kinases (SFKs). Recently, we showed NMT2 expression is lost in numerous haematological cancer cell lines (including AML) and that these haematological cancer cell lines are exquisitely sensitive to the pan-NMT inhibitor PCLX-001. PCLX-001 recently entered human clinical trials as once daily oral therapy for relapsed/refractory B-cell Non-Hodgkin Lymphoma and advanced solid malignances. Dysregulation and oncogenic activity of SFKs occurs frequently in AML, suggesting NMT inhibition could provide therapeutic benefit in this indication. Data analysis from the TCGA transcriptome database revealed that high NMT1 and low NMT2 were associated with reduced overall and event-free survival in adult AML. Moreover, high NMT1 - but not NMT2 - expression is associated with proliferative gene sets in AML cell lines. AML cell lines treated with PCLX-001 showed a significant reduction in total protein myristoylation, reduced levels of SFK proteins and SFK phosphorylation as well as significant increases in ER stress marker BIP protein and caspase 3 cleavage. PCLX-001 induced apoptosis in AML cell lines and patient blasts at concentrations that spared a large proportion of peripheral blood lymphocytes and monocytes from healthy individuals. PCLX-001 monotherapy had dose-dependent anticancer activity in an AML MV-4-11 cell line derived xenograft (CDX) and two AML patient derived xenografts (PDXs) and produced complete remissions in subcutaneous AML CDX. In tail-vein injected PDX models, PCLX-001 treatment resulted in up to 95% reduction of human CD45+ cells in peripheral blood and bone marrow. PCLX-001 preferentially targeted AML cells inducing apoptosis and reducing leukemic burden. These findings validate NMT inhibition as a novel therapeutic strategy for AML and warrant the evaluation of PCLX-001 in clinical trials for adult AML. Citation Format: Jay Gamma, Aishwarya Iyer, Megan Yap, Zoulika Zak, Krista Vincent, Cassidy Ekstrom, Qiang Liu, Erwan Beauchamp, Lynne Postovit, Jean Wang, John R. Mackey, Naveen Pemmaraju, Gautam Borthakur, Joseph Brandwein, Luc Berthiaume. Targeting N-myristoylation for therapy of adult acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5662.

Abstract 610: The expression of the PVRIG/TIGIT pathway is dominant in the bone marrow of patients with multiple myeloma

Abstract Blocking inhibitory immune receptors has shown limited clinical benefit in patients with multiple myeloma (MM), warranting the discovery of alternative immune inhibitory pathways. Blockade of the immune checkpoint TIGIT was shown to enhance anti-tumor immunity in MM pre-clinical models. In addition to TIGIT, the DNAM1 axis includes the novel inhibitory receptor PVRIG. Both TIGIT and PVRIG deliver inhibitory signals to T and NK cells and compete with the co-activating receptor DNAM1 for binding to PVR and PVRL2, respectively. Accordingly, TIGIT and PVRIG co-blockade was shown to synergize in enhancing anti-tumor immunity in preclinical models. PVRL2 and PVR were shown to be expressed on MM plasma cells in the bone marrow (BM). In this study, we evaluated DNAM1 axis receptor expression in the BM of patients with MM. BM mononuclear cells derived from 21 patients with MM were analyzed for the expression of PD1 and DNAM1 axis molecules by flow cytometry. Patients classified with progressed disease (PD), or complete response (CR) were treated with multiple lines of therapies including targeted therapies, chemotherapies, proteasome inhibitors. PVRIG demonstrated the highest expression among all evaluated receptors on NK (88%), NKT (81%) and CD8+ T cells (79%), significantly higher than PD1 (p&amp;lt;0.0001) and TIGIT (P&amp;lt;0.001). TIGIT showed substantially increased expression on NK (64%, P&amp;lt;0.0001) and CD8+ T cells (58%, p&amp;lt;0.05) compared to PD1 (expressed on 12% of NK cells and 42% of CD8+ T cells). Importantly, 50% and 60% of CD8+ and NK cells, respectively, co-expressed TIGIT and PVRIG, and all examined cell populations showed increased levels of DNAM1 (&amp;gt;50%). Additionally, the expression of PVRIG ligand, PVRL2, on BM plasma cells from 6 patients with MM was demonstrated by immuno-histochemistry and flow cytometry. A fraction of CD8+ T cells were DNAM1- and positive for PVRIG/TIGIT/PD1 (22%), suggesting the accumulation of an exhausted CD8+ T cell population in the MM tumor microenvironment. PVRIG showed significantly higher (p&amp;lt;0.0001) expression on DNAM+ CD8+ T cells (81%), compared to TIGIT (43%) and PD1 (34%), supporting the potential of PVRIG blockade to enhance DNAM1 signaling and subsequent CD8+ T cell activation. Finally, patients with CR had a trend towards higher DNAM1 expression on CD8+ T cells (75%) compared to PD patients (54%, p=0.057). To conclude, DNAM1 axis receptors are dominantly expressed on MM BM lymphocytes, with PVRIG exhibiting the most prominent expression. The reduced expression of DNAM1 in patients with PD suggests a link between DNAM1 axis and clinical outcome. Our findings highlight for the first time the dominant expression of PVRIG, as well as TIGIT and suggest that combined blockade of these checkpoints may potentially benefit patients with MM, placing the DNAM1 axis as a promising therapeutic pathway in MM therapy. Citation Format: Masha Frenkel, Zoya Alteber, Ning Xu, Mingjie Li, Haiming Chen, Gady Cojocaru, James Berenson, Eran Ophir. The expression of the PVRIG/TIGIT pathway is dominant in the bone marrow of patients with multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 610.