Transcriptional Characterization of Myelofibrotic Bone Marrow Microenvironment Reveals Distinct Tumor Microenvironment in JAK2+ and Calr+ PMF Marrows

Abstract Background: Primary myelofibrosis (PMF) is a clonal stem cell disorder associated with somatic mutations in three genes: Janus kinase 2 (JAK2), calreticulin (CALR) and thrombopoietin receptor (MPL). Although, our understanding of the microenvironment in PMF is limited, in PMF levels of Treg, cytotoxic T-cells, B-cells, macrophages and megakaryocyte cell populations have been reported to be elevated in either peripheral blood or bone marrow (BM) (Barosi Curr Hematol Malig Rep 2014). In addition, various cellular pathways including JAK/STAT, TGFβ1, and cytokine pathways (CXC family, hematopoietin family, PDGF family and TGF family), have been reported to play an important role in the dysregulation of hematopoietic cell proliferation and disease progression. Here-in we characterize the tumor microenvironment in formalin fixed paraffin embedded (FFPE) BM biopsies obtained from PMF patients and correlate these findings with mutational status. Methods: We applied the enzyme-free NanoString nCounter® PanCancer Immune Profiling Panel system (NanoString Technologies, Inc., Seattle, WA) to identify and assess immunological function in the microenvironement of archival FFPE bone marrow samples from patients with PMF. Twelve archival bone marrow FFPE biopsies from PMF patients along with clinical information and 5 normal controls were analyzed using upto 500ng of RNA (at 100ng/ul) for digital expression profiling. The panel included 109 genes that define 24 immune cell types and populations and forty housekeeping genes that facilitate sample-to-sample normalization. Data analysis was performed using nSolver software 3.0 and the Advanced Analysis Module (v.1.0.84). Results: Gene expression profiles for cellular immune pathways were analyzed for global changes based mutation. Globally, cellular functions involving immune cell development and cellular responses/functions were dramatically decreased in myelofibrotic marrow (chemokines, complement, cytokines, cytotoxicity) when compared to normal marrow. However, only in areas of adhesion, antigen processing, transporter function and senescence genes were transcription levels elevated over normal controls. Differential expression analysis of JAK2V617F+ marrow showed decreased expression of genes involved in cell regulation, NK cell function, T-cell functions and pathogen defense and increased expression of genes involved in inflammation, chemokines and transporter functions over normal marrow. Whereas CALR+ bone marrow biopsies showed fewer genes down regulated and an increased number of genes up regulated, particularly involved in fibrosis, inflammation, chemokines, adhesion, antigen processing and regulation. Pathway analysis suggested a particular role for FLT3 ligand in myeloid stem cell regulation, thrombospondin (THBS1) which has been reported to promote the activation of the latent forms of TGFβ1, and mitogen-activated protein kinases (JNK1, ERK) in PMF cell proliferation and differentiation. Conclusions: Digital immune expression profiling reveals a distinct PMF tumor microenvironment and illustrates potential transcriptional differences based on their mutational status. (JAK2+ or CALR+). These transcriptional changes in myelofibrotic marrow are reflected in global changes in immune cells and pathway activation These data provide for the first time in situ evidence of the importance of the immune system in PMF pathogenesis. Barosi G, 2014 An immune dysregulation in MPN. Curr Hematol Malig Rep 9:331-339. Disclosures No relevant conflicts of interest to declare.

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A pan-cancer study of selenoprotein genes as promising targets for cancer therapy

BMC Medical Genomics ◽

10.1186/s12920-021-00930-1 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Wentao Wu ◽

Daning Li ◽

Xiaojie Feng ◽

Fanfan Zhao ◽

Chengzhuo Li ◽

...

Keyword(s):

Drug Resistance ◽

Tumor Microenvironment ◽

Drug Sensitivity ◽

Immune Cell ◽

Expression Profiles ◽

Health Benefit ◽

Glutathione Peroxidases ◽

Expression Of Genes ◽

Important Health ◽

And Control

Abstract Background The most important health benefit of selenium (Se) is in the prevention and control of cancer. Glutathione peroxidases (GPXs) and thioredoxin reductases (TXNRDs) are selenoenzymes that are thought to play a role in oxidative stress. The differential expression of genes of the TXNRD and GPX families is closely related to carcinogenesis and the occurrence of cancer. This study comprehensively analyzed the expression profiles of seven genes in the TXNRD and GPX families, in terms of their correlations with patient survival and immune-cell subtypes, tumor microenvironment, and drug sensitivity. Results The expression profiles of genes in the TXNRD and GPX families differ between different types of cancer, and also between and within individual cancer cases. The expression levels of the seven analyzed genes are related to the overall survival of patients. The TXNRD1 and TXNRD3 genes are mainly related to poor prognoses, while other genes are related to good or poor prognoses depending on the type of cancer. All of the genes were found to be correlated to varying degrees with immune-cell subtypes, level of mechanistic cell infiltration, and tumor cell stemness. The TXNRD1, GPX1, and GPX2 genes may exert dual effects in tumor mutagenesis and development, while the TXNRD1, GPX1, GPX2, and GPX3 genes were found to be related to drug sensitivity or the formation of drug resistance. Conclusions The results will greatly help in identifying the association between genes and tumorigenesis, especially in the immune response, tumor microenvironment, and drug resistance, and very important when attempting to identify new therapeutic targets.

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603 Targeted STAT3 degradation leads to remodeling of an immunosuppressive tumor microenvironment and subsequent sensitization to immune checkpoint therapy

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.603 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A633-A633

Author(s):

Joyoti Dey ◽

Phillip Liu ◽

Michele Mayo ◽

Rahul Karnik ◽

Bin Yang ◽

...

Keyword(s):

Gene Expression ◽

T Cell ◽

Tumor Microenvironment ◽

Gene Expression Profiling ◽

Cancer Cell ◽

Immune Checkpoint ◽

Expression Profiling ◽

Immune Cell ◽

B Cell Lymphoma ◽

Activation Markers

BackgroundSignal Transducer and Activator of Transcription 3 (STAT3), a multifaceted transcription factor, is aberrantly activated across a variety of malignancies; however, its selective targeting has to-date remained a therapeutic challenge. STAT3 plays a pivotal role in shaping the tumor immune landscape through cancer cell-intrinsic mechanisms, direct regulation of immune cell function and via cancer cell- tumor microenvironment (TME) crosstalk, that collectively result in an immunosuppressive TME. Targeted protein degradation represents a novel therapeutic modality enabling direct targeting of previously undruggable oncoproteins. We have developed potent and selective STAT3 heterobifunctional degraders demonstrating activity across diverse tumor and immune cell types.MethodsWe investigated the immunomodulatory impact of STAT3 degradation on tumorigenesis in syngeneic mouse models representing cancers with heterogeneous immune milieus. Methods included in vivo pharmacological approaches, immunophenotyping and gene expression profiling.ResultsTreatment of CT-26 (colorectal cancer) and A20 (B-cell lymphoma) tumor-bearing mice with a STAT3 degrader resulted in significant tumor growth inhibition compared to controls, with loss of STAT3 protein in both tumor cells and TME. This was accompanied by a decrease in M2 polarized macrophages and concomitant increases in M1 polarized macrophages and tumor infiltrating lymphocytes. The anti-tumor responses were abrogated by antibody mediated CD8+ T cell depletion or by using immunodeficient host-strains implicating the observed efficacy to be predominantly driven by immune-directed mechanisms. Gene expression profiling of STAT3 degrader-treated CT-26 tumors showed marked increases in proinflammatory genes including T cell and M1 macrophage activation markers, compared to controls. Notably, induction of an Ifnγ-responsive gene signature (Ifnγ, Stat1, Cxcl9, Cxcl10, Ido1) suggested that STAT3 degradation results in a T-cell inflamed phenotype associated with responsiveness to immune checkpoint therapy (ICT). Furthermore, on-treatment tumors showed an upregulation of genes such as Pdl1, Ctla4, Lag3 which reflect T cell activation as well as counterregulatory mechanisms. Therefore, we evaluated STAT3 degradation in combination with anti-PD1 in these models which are poorly responsive to anti-PD1 monotherapy. Robust synergy was observed in the CT-26 model with 60% complete responses and development of immunological memory as confirmed by tumor re-challenge studies. Studies are underway to ascertain the applicability of this combination therapy in different tumor-immune contextures and indications, and to elucidate the mechanistic basis of synergy.ConclusionsSTAT3 degradation remodels an immunosuppressed TME activating anti-tumor immunity as monotherapy and effectively combines with anti-PD1. These data provide a rationale for selectively degrading STAT3 as a strategy to sensitize cancers with relevant immune contextures to ICT in the clinic.

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Expansion and Characterization of Iovance Marrow Infiltrating Lymphocytes: A Potential Novel Therapeutic Strategy for the Treatment of Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-114825 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5683-5683

Author(s):

Lavakumar Karyampudi ◽

Ian Frank ◽

Michelle Blaskovich ◽

John C. Byrd ◽

Cecile Chartier ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Tumor Microenvironment ◽

Mononuclear Cells ◽

Immune Cell ◽

T Cell Subsets ◽

Bone Marrow Mononuclear Cells ◽

Equity Ownership ◽

Cell Fractions ◽

Infiltrating Lymphocytes

Abstract Background: Acute myeloid leukemia (AML) is a poor-prognosis malignancy arising from hematopoietic stem/progenitor cells. To date, novel immunotherapies such as checkpoint inhibitors, vaccines and adoptive cell therapy (ACT) using CAR T cells have demonstrated only modest success for the treatment of patients who are ineligible for marrow transplantation and have minimal residual disease; additional approaches are warranted (Beyar-Katz O and Gill S, Clin Cancer Res 2018). ACT with tumor infiltrating lymphocytes (TIL) has emerged as an effective treatment for patients with metastatic melanoma (Goff SL et al, J Clin Oncol 2016), likely owing to the heterogeneous population of tumor-reactive T cells that comprises the TIL products. As demonstrated for solid cancers, such tumor-reactive T cells are preferentially found in the tumor microenvironment (Gros A et al JCI 2014; Thommen DS et al Nat Med 2018). By avoiding the highly immunosuppressive tumor microenvironment, ex vivo activation of those cells rescues them from tolerance and anergic status. We hypothesized that, in the case of AML for which the bone marrow represents the tumor microenvironment, tumor antigen-specific T cells could be recovered from the patient bone marrow to produce a highly effective therapeutic product that is cytotoxic to AML tumor cells. We present findings related to the ex vivo expansion of Iovance marrow infiltrating lymphocytes (MIL) for the treatment of AML patients. Methods: Immune cell and non-immune cell fractions were sorted from bone marrow mononuclear cells. Immune cell fractions loaded with sonicated non-immune cell fractions were expanded for 14 days in the presence of αCD3/αCD28 beads and interleukin-2 (IL-2) to generate MIL products. Phenotypic and functional characteristics of the cells were determined by flow cytometry and enzyme-linked immunospot assay (ELISpot). Results: MIL were generated from isolated bone marrow mononuclear cells (n=2) with a mean expansion fold of 86 (range 78-93). Equal percentage of CD4+ and CD8+ T cell subsets constituted the MIL products. Phenotypic analysis of the cells showed that the majority of T cell subsets are effector memory and CD28 positive. Low percentages of the T cell subsets were positive for immunosuppressive markers PD-1 and LAG3. ELISpot analysis demonstrated that MIL were readily activatable and produced normal levels of IFNγ in response to CD3/CD28 stimulation. Antigen specificity of MIL is being investigated. Conclusion: We demonstrated the feasibility of MIL expansion from bone marrow mononuclear cells from AML patients. MIL are functionally active and mostly comprised of effector memory T cells. Confirmation of tumor cell antigen specificity will determine whether MIL may deploy a robust anti-tumor activity in vivo. Disclosures Karyampudi: Iovance Biotherapeutics: Employment, Equity Ownership. Frank:Iovance Biotherapeutics: Employment, Equity Ownership. Blaskovich:Iovance Biotherapeutics: Equity Ownership. Chartier:Iovance Biotherapeutics: Equity Ownership.

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IMMU-02. GENOMIC AND TRANSCRIPTOMIC CORRELATES OF IMMUNOTHERAPY RESPONSE WITHIN THE TUMOR MICROENVIRONMENT OF LEPTOMENINGEAL METASTASES

Neuro-Oncology ◽

10.1093/neuonc/noab196.362 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi92-vi92

Author(s):

Christopher Alvarez-Breckenridge ◽

Sanjay Prakadan ◽

Samuel Markson ◽

Albert Kim ◽

Naema Nayyar ◽

...

Keyword(s):

Tumor Microenvironment ◽

Single Cell ◽

T Cell Activation ◽

Antigen Processing ◽

Cell Activation ◽

Immune Cell ◽

Checkpoint Inhibitors ◽

Leptomeningeal Disease ◽

Ifn Gamma ◽

Molecular Features

Abstract Leptomeningeal disease (LMD) is a devastating complication of solid tumor malignancies, with dire prognosis and no effective systemic treatment options. Over the past decade, the incidence of LMD has steadily increased due to therapeutics that have extended the survival of cancer patients, highlighting the need for new interventions. To examine the efficacy of immune checkpoint inhibitors (ICI) in patients with LMD, we completed two phase II clinical trials utilizing either Pembrolizumab alone or the combination of Ipilimumab and Nivolumab. We investigated the cellular and molecular features underpinning observed patient trajectories in these trials by applying single-cell RNA and cell-free DNA profiling to longitudinal cerebrospinal fluid (CSF) draws from enrolled patients. We isolated and sequenced 34,742 cells from both the malignant and immune compartment within CSF. Amongst the 19 patients included in the cohort, there were 13 pre-treatment and 24 post-treatment samples, and 9 patients were sampled across multiple timepoints. We detected dynamic changes in immune cell recruitment into the CSF and activation within 30 days of ICI, including increased effector T cell activation and IFN-gamma response pathways within T cells. Moreover, the overall level of IFN-gamma response and antigen processing within 30 days of ICI in malignant cells correlated with survival past clinical trial primary endpoint. Lastly, we observed evidence of longitudinal outgrowth of distinct immunogenic clones over the course of ICI. Overall, our study describes the liquid LMD tumor microenvironment prior to and following ICI treatment and provides unique insights into the compartmental and temporal variation during the course of ICI. Moreover, our findings demonstrate the clinical utility of cell- free and single-cell genomic measurements for LMD research.

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Gene Expression Profiling of the Bone Marrow Mononuclear Cells from Patients with Melodysplastic Syndrome.

Blood ◽

10.1182/blood.v104.11.3429.3429 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3429-3429

Author(s):

Zi-xing Chen ◽

Jun Qian ◽

Wei Wang ◽

Jian-nong Cen ◽

Yong-quang Xue

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Gene Expression Profiling ◽

Expression Pattern ◽

Expression Profiling ◽

Cdna Clones ◽

Rt Pcr ◽

Transcript Levels ◽

Significant Difference ◽

Normal Controls

Abstract Myelodysplastic syndrome (MDS), a clonal disorder of hematopoietic stem cells, is characterized by cytopenia in at least one lineage of peripheral blood and dysplastic features in BM cells. It has been considered as a preleukemic condition for a high risk of transformation to AML. The FAB classification has divided MDS into five clinical categories: RA, RAS, RAEB, RAEB-t and CMML. To date, the key genes involved in the pathogenesis and its progression to acute leukemia has not been clearly identified. This was at least partially caused by a narrow scale of genes which could only been studied once a time by using traditional methods. The present study was to investigate the molecular mechanism of MDS by using cDNA microarray techniques. We have first applied total RNA of bone marrow monoculear cells (BMNCs) from 2 MDS patients (one RA and one RAS) to a BioStar H141 microarray ( United Gene Holdings Co. LTD, Shanghai, China) containing 13484 gene cDNA clones and ets. Based on the preliminary results of these assay, 500 genes which were shown most remarkably differentially expressed in MDS compare to normal control, and with known functions and potential involvement in hematopoiesis regulation, were selected to design and compose 10 cDNA chips. These arrays were then used to analyze the gene expression pattern of BMNCs from 10 patients with MDS, including 4 RA, 1 RTC, 4 RAEB, and 1 RAEBt. To confirm the microarray results and to evaluate the disease relevance of some selected genes from array results, a semiquantitative RT-PCR was performed to analyze gene expression in fifty addi-tional patients with MDS (28 RA, 15 RAEB, 7 RAEBt), 38 acute myeloid leukemia (7 M1, 12 M2, 4 M3, 4 M4, 5 M5, 6 M6), 15 atypical anaplastic anemia (AA), and 12 hypercellular anemia. Our results revealed that 95 genes were abnormally expressed in at least five MDS patients compared to normal controls, involving cell growth and differentiation regulation, cell cycle control, signaling, redox, such as thrombospondin 1 (THBS1), phosphatase and tensin homolog (PTEN), MAD, DNA-damage-inducible transcript 3 (DDIT3), ets variant gene 1 (ETV1), and G1 to S phase transition 1 (GSPT1). These MDS patients in different risk groups could be clustered into two groups overall by hierarchical clustering, wherein a case with isolated thrombocytopenia and other RA patients were clustered into two subgroups 5 genes (RNAHP, DDIT3, FOXO3A, GSPT1, and ETV1) which displayed a most marked differential expression pattern in most MDS patients, were selected as "candidate genes". Consistent expression patterns of 3 (60%) in 5 genes were confirmed by semiquantitative RT-PCR. Further analysis showed different transcript levels of RNAHP, DDIT3 among patients with MDS in different risk group, AML, and normal controls. Meanwhile, the transcript levels of five genes were also compared in the patients with RA, AA and other hypercelluler anemia patients. There was significant difference in RNAHP levels between RA and CAA, or other hypercelluler anemia (P<0.05), similar diversity also seen in ETV1 levels between RA and AA (P<0.01). However, significant differences in DDIT3, FOXO3A, and GSPT1 levels were not observed. Our study suggested that gene expression profiling of MDS patients may reveal a specific transcription patterns for BMNCs in MDS. The abnormal expression of RNAHP, DDIT3 and ETV1 may play roles in the patho-genesis of MDS and may provide useful biomarkers for the molecular diagnosis of MDS.

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Effect of miR-124 on the Biological Characteristics of Bone Marrow Mesenchymal Stem Cells in Tumor Microenvironment

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2020.2235 ◽

2020 ◽

Vol 10 (2) ◽

pp. 151-156

Author(s):

Linfu He ◽

XiaoyanHe ◽

Xiaocui Liu ◽

Wenjing Shi ◽

PeiXu ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Cell Proliferation ◽

Mesenchymal Stem Cells ◽

Tumor Microenvironment ◽

Glioma Cells ◽

Biological Characteristics ◽

Brain Glioma ◽

Marrow Mesenchymal Stem Cells

miR-124 affects migration and differentiation of mesenchymal stem cells (MSCs), but its role in bone marrow mesenchymal stem cells (BMSCs) in tumor microenvironment remain unclear. Therefore, our study aims to assess miR-124’s role in BMSCs in glioma microenvironment. BMSCs were isolated and co-cultured with glioma cells using Transwell chamber and then transfected with miR-124 mimic/NC followed by analysis of BMSCs biological characteristics (cell proliferation by CCK8, miR-124 level by Real-time PCR, cell cycle and apoptosis by flow cytometry, and cell migration by Transwell). There were significant differences of cell proliferation, migration, apoptosis and cell cycle in BMSCs group compared to co-culture and co-culture+ miR-NC group (P < 0.05), without no difference compared to co-culture + miR-124 group. In addition, co-culture + miR-124 also showed significant differences of BMSCs' biological characteristics (P < 0.05). miR-124 can alter the biological characteristics of BMSCs in the tumor microenvironment simulated by C6 brain glioma cells.

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Skeletal unloading causes organ-specific changes in immune cell responses

Journal of Applied Physiology ◽

10.1152/jappl.1993.75.6.2734 ◽

1993 ◽

Vol 75 (6) ◽

pp. 2734-2739 ◽

Cited By ~ 35

Author(s):

J. W. Armstrong ◽

K. A. Nelson ◽

S. J. Simske ◽

M. W. Luttges ◽

J. J. Iandolo ◽

...

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

Immune System ◽

T Cell ◽

Lymph Nodes ◽

Immune Cell ◽

Killer Cell ◽

T Cell Proliferation ◽

Skeletal Unloading ◽

Natural Killer Cell Cytotoxicity

The effects of skeletal unloading using antiorthostatic tail suspension on the mouse immune system are tissue specific. This phenomenon was demonstrated by analyzing cells from the lymph nodes, spleen, and bone marrow. Phytohemagglutinin-induced T-cell proliferation was depressed in lymph nodes after 11 days of antiorthostatic suspension. In contrast, splenic T-cell proliferation in response to phytohemagglutinin was enhanced. Splenic natural killer cell cytotoxicity was unchanged after suspension, which demonstrated the organ- and cell-specific effects of skeletal unloading. Whereas antiorthostatic suspension induced minimal changes in bone, there was a significant depression in the number of macrophage precursors in the bone marrow. Overall, skeletally unloaded animals had slightly higher blood corticosterone levels than did control animals; however, it did not appear to be responsible for the observed changes. In conclusion, skeletal unloading produces organ- and cell-specific changes in the murine immune system rather than a generalized immunosuppression.

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Deciphering the immunosuppressive tumor microenvironment in ALK- and EGFR-positive lung adenocarcinoma

Cancer Immunology Immunotherapy ◽

10.1007/s00262-021-02981-w ◽

2021 ◽

Author(s):

Jan Budczies ◽

Martina Kirchner ◽

Klaus Kluck ◽

Daniel Kazdal ◽

Julia Glade ◽

...

Keyword(s):

Gene Expression ◽

Tumor Microenvironment ◽

Lung Adenocarcinoma ◽

Expression Profiling ◽

Immune Cell ◽

Expression Profiles ◽

Immune Therapy ◽

Immune Reactivity ◽

Immune Gene ◽

Alk Positive

Abstract Introduction The advent of immune checkpoint blockade (ICB) has led to significantly improved disease outcome in lung adenocarcinoma (ADC), but response of ALK/EGFR-positive tumors to immune therapy is limited. The underlying immune biology is incompletely understood. Methods We performed comparative mRNA expression profiling of 31 ALK-positive, 40 EGFR-positive and 43 ALK/EGFR-negative lung ADC focused on immune gene expression. The presence and levels of tumor infiltration lymphocytes (TILs) as well as fourteen specific immune cell populations were estimated from the gene expression profiles. Results While total TILs were not lower in ALK-positive and EGFR-positive tumors compared to ALK/EGFR-negative tumors, specific immunosuppressive characteristics were detected in both subgroups: In ALK-positive tumors, regulatory T cells were significantly higher compared to EGFR-positive (fold change: FC = 1.9, p = 0.0013) and ALK/EGFR-negative tumors (FC = 2.1, p = 0.00047). In EGFR-positive tumors, cytotoxic cells were significantly lower compared to ALK-positive (FC = − 1.7, p = 0.016) and to ALK/EGFR-negative tumors (FC = − 2.1, p = 2.0E-05). A total number of 289 genes, 40 part of cytokine–cytokine receptor signaling, were differentially expressed between the three subgroups. Among the latter, five genes were differently expressed in both ALK-positive and EGFR-positive tumors, while twelve genes showed differential expression solely in ALK-positive tumors and eleven genes solely in EGFR-positive tumors. Conclusion Targeted gene expression profiling is a promising tool to read out tumor microenvironment characteristics from routine diagnostic lung cancer biopsies. Significant immune reactivity including specific immunosuppressive characteristics in ALK- and EGFR-positive lung ADC, but not a total absence of immune infiltration supports further clinical evaluation of immune-modulators as partners of ICB in such tumors.

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Eosinophils in the Bone Marrow Microenvironment: Effects On Malignant Plasma Cell Biology.

Blood ◽

10.1182/blood.v120.21.2917.2917 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2917-2917

Author(s):

Tina W. Wong ◽

Denise K. Walters ◽

Hirohito Kita ◽

Diane F. Jelinek

Keyword(s):

Bone Marrow ◽

Biological Activity ◽

Tumor Microenvironment ◽

Disease Progression ◽

Cell Biology ◽

Immune Cell ◽

Conflicts Of Interest ◽

Immune Defense ◽

Common Source ◽

Cell Clusters

Abstract Abstract 2917 Multiple myeloma (MM) is a cancer in the bone marrow (BM) characterized by the accumulation of transformed plasma cells (PCs). The pre-malignant form of the disease, monoclonal gammopathy of undetermined significance (MGUS), shares many of the genetic abnormalities found in MM, including chromosomal translocations, hyperdiploidy, and gene-specific mutations. Given this, we believe other factors within the tumor microenvironment must contribute to disease progression by influencing cell survival and/or proliferation. Eosinophils (Eos) are granulocytic leukocytes that are best known for their involvement in host immune defense and pathologic states such as allergy and asthma. Recently, they were also shown to play a role in the regulation of murine BM PC homeostasis via their secretion of IL-6 and APRIL. Murine BM PCs and Eos both express CXCR4 and are believed to home to CXCL12 expressing stromal cells (SCs) in the BM. The goal of this study, therefore, was to investigate whether Eos, or soluble mediators released by Eos, have biological activity on MM cells. Thus, it is possible that this type of innate immune cell may be present in the tumor microenvironment in MM patients to support disease progression. To our knowledge, a potential role for Eos in MM has not been previously studied. We began our studies by assessing whether Eos are co-localized with normal BM PCs and/or MM PCs. Immunofluorescence analysis of BM core biopsies from normal subjects revealed occasional colocalization of PCs with Eos. Similarly, MM biopsies showed regions of MM cell clusters with increased Eos density, suggesting possible biological interactions. However, we also observed regions of MM cell clusters that lack Eos, which could indicate the liberation of these transformed cells from the requirement of Eos for survival/proliferation. Next, using Eos isolated from human BM aspirates and a panel of disease-relevant human MM cell lines (HMCLs) extensively characterized in our laboratory, we aimed to verify that MM cells and Eos could both migrate toward the chemokine CXCL12. Our data showed that indeed the KAS-6/1, ANBL-6, DP-6, and KP-6 HMCLs and human BM Eos all migrated toward this chemokine. We then determined if Eos had biological activity towards MM cells as revealed by enhanced DNA synthesis. Consistent with our interpretation of the immunofluorescence stained sections, the proliferation of some, but not all, HMCLs was enhanced when cocultured with Eos isolated from either human BM or peripheral blood. To address whether contact between Eos and HMCLs was required for this phenomenon, we assessed HMCL proliferation upon treatment with Eos culture supernatant (SN). Our data suggest that the effect of Eos on the Eos-inducible HMCLs can be contact-independent as treatment of these HMCLs with Eos SN increased their proliferation. Similarly, proliferation of primary CD138+ MM cells was also enhanced when treated with Eos SN. Because BM PCs reside in niches that include support cells such as SCs and that proliferation of MM PCs is enhanced in the presence of SCs, we next questioned whether Eos can substitute for SCs in this niche or if Eos and SCs support PC survival/proliferation through different mechanisms. We observed that Eos and BM SCs together stimulated more HMCL proliferation than either cell type did alone, indicating the presence of non-redundant roles for the two cell types. Finally, we began to investigate the mechanism by which Eos enhance HMCL proliferation. In contrast to prior reports that murine Eos express IL-6, mRNA transcripts of IL-6, a known proliferation-inducing cytokine for MM cells, were not detected in human Eos. Moreover, analysis of the Eos SN showed an absence of IL-6. Neutralization of IL-6 in the HMCL-Eos coculture did not abolish the induced proliferation. Altogether, these data suggest that human Eos utilize a yet to be identified, IL-6-independent mechanism to support malignant PC proliferation. Taken together, our data show that Eos and MM cells can colocalize in the BM via their migration toward a common source of CXCL12, i.e. BM SCs, to create a niche that promotes tumor cell growth. Eos can enhance malignant PC proliferation via soluble products, although additional contact-dependent effects may exist as well and have not been explored. Additional studies are currently underway to further characterize the mechanism by which Eos may influence the biology of MM in the tumor microenvironment. Disclosures: No relevant conflicts of interest to declare.

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Single Cell Transcriptome Profile of Myeloma and Immune Cell Characteristics in Patients with Durable Response Post CART

Blood ◽

10.1182/blood-2021-153254 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3838-3838

Author(s):

Yan Asmann ◽

Ying Li ◽

Taxiarchis Kourelis ◽

Moritz Binder ◽

Wilson I Gonsalves ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

Immune Cell ◽

Advisory Committees ◽

Durable Response ◽

Myeloma Cells ◽

Expression Of Genes ◽

Car T ◽

Differential Gene

Abstract Background: Chimeric antigen receptor T cell therapy (CAR-T) is a recent therapeutic advance for the treatment of myeloma. Among the FDA approved and investigational CAR-T, primary refractory disease post CAR-T infusion is uncommon, however most patients eventually relapse. To gain insight to the myeloma cell and immune cell characteristics associated with early relapse (PD) versus durable response (DR), we examined myeloma cells and immune cells in both the bone marrow and blood by single cell RNA-seq. Method: Bone marrow aspirate and blood samples were collected prior to BCMA targeted CAR-T therapy. CD138 + and CD138 neg cells from bone marrow (BM) and peripheral blood mononuclear cells (PBMNC) were collected for 5' based scRNA-seq (10X Genomics). CD138 + cells were clustered by Harmony integration. Top expressing genes in unique clusters for PD and DR were analyzed by Ingenuity Pathway Analyses. To profile CD138 neg BM cells and PBMNC from myeloma patients, the scRNA-seq dataset was mapped to the human PBMC reference using the Seurat v4 reference-guiding approach. Differential gene expression between responders and non-responders was performed using the FindMarkers function for each of the immune cell clusters in the BM and PB. Results: BM and PB samples from 15 patients were analyzed. Among these, 5 patients had relapsed disease more than 1-year post CAR-T infusion (Durable Response, DR), and 10 patients had relapse within 1 year (early relapse, PD). Among these, CD138+ cells were available from 3 DR and 4 PD for analysis. Multimodal expression of BCMA were seen among the CD138+ cells across all samples. Using the mode of the highest expression level as a cut-off, patients with DR had a trend toward having higher fraction of BCMA high CD138+ cells compared to pts with PD. Clustering of all CD138+ cells by Harmony integration identified 12 distinct clusters, among which two clusters were unique to pts in DR, and one was unique to patients in PD. Ingenuity pathway analysis identified the top marker genes in these 3 clusters to be enriched in pathways for IL-15 signaling, BCR signaling, and primary immunodeficiency signaling. (Figure 1A-E.) Differential gene expression for immune cell clusters from CD138 neg BM cells and PBMNC were compared between pts in PD and DR. Interestingly, different patterns were seen in immune cells in PB and BM (Figure 1G). For example, compared to pts in PD, those with DR had decreased signaling in CD16 PB monocytes for interferon signaling and differentiation to macrophages, whereas the CD16 monocytes in the BM in pts in DR had increased expression of mitochondrial genes for anti-oxidant stress, polarization to M1 macrophage and anti-apoptosis. Similarly, pts in DR had CD8 Tcm in the BM with increased expression of genes involved in cell adhesion and anti-apoptosis, and CD4 Tcm in the BM with increased expression of genes in protein metabolism. Conclusion: scRNAseq analysis of myeloma cells suggest different tumor transcriptome profile may be identified in myeloma cells that could relapse early after CAR-T therapy. In addition, host immune profile both in the BM microenvironment and in systemic PB circulation could be associated with durable clinical response. Figure 1 Figure 1. Disclosures Kapoor: Pharmacyclics: Consultancy; Glaxo SmithKline: Research Funding; Amgen: Research Funding; Sanofi: Research Funding; AbbVie: Research Funding; Ichnos Sciences: Research Funding; Sanofi: Consultancy; Regeneron Pharmaceuticals: Research Funding; Takeda: Research Funding; Karyopharm: Research Funding; Karyopharm: Consultancy; Cellectar: Consultancy; BeiGene: Consultancy. Dingli: GSK: Consultancy; Janssen: Consultancy; Sanofi: Consultancy; Novartis: Research Funding; Apellis: Consultancy; Alexion: Consultancy. Kumar: Bluebird Bio: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Novartis: Research Funding; BMS: Consultancy, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche-Genentech: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Antengene: Consultancy, Honoraria; Carsgen: Research Funding; Oncopeptides: Consultancy; Beigene: Consultancy; Amgen: Consultancy, Research Funding; Merck: Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding. Lin: Merck: Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; Takeda: Research Funding; Bluebird Bio: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Sorrento: Consultancy; Juno: Consultancy; Legend: Consultancy; Gamida Cell: Consultancy; Vineti: Consultancy; Novartis: Consultancy; Janssen: Consultancy, Research Funding.

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