Preclinical models for prediction of immunotherapy outcomes and immune evasion mechanisms in genetically heterogeneous multiple myeloma

The historical lack of preclinical models reflecting the genetic heterogeneity of multiple myeloma (MM) hampers the advance of therapeutic discoveries. To circumvent this limitation, we screened mice engineered to carry eight MM lesions (NF-κB, KRAS, MYC, TP53, BCL2, cyclin D1, MMSET/NSD2 and c-MAF) combinatorially activated in B lymphocytes following T cell-driven immunization. Fifteen genetically diverse models developed bone marrow (BM) tumors fulfilling MM pathogenesis. Integrative analyses of ∼500 mice and ∼1,000 patients revealed a common MAPK-MYC genetic pathway that accelerated time to progression from precursor states across genetically heterogeneous MM. MYC-dependent time to progression conditioned immune evasion mechanisms that remodeled the BM microenvironment differently. Rapid MYC-driven progressors exhibited a high number of activated/exhausted CD8+ T cells with reduced immunosuppressive regulatory T (Treg) cells, while late MYC acquisition in slow progressors was associated with lower CD8+ T cell infiltration and more abundant Treg cells. Single-cell transcriptomics and functional assays defined a high ratio of CD8+ T cells versus Treg cells as a predictor of response to immune checkpoint blockade (ICB). In clinical series, high CD8+ T/Treg cell ratios underlie early progression in untreated smoldering MM, and correlated with early relapse in newly diagnosed patients with MM under Len/Dex therapy. In ICB-refractory MM models, increasing CD8+ T cell cytotoxicity or depleting Treg cells reversed immunotherapy resistance and yielded prolonged MM control. Our experimental models enable the correlation of MM genetic and immunological traits with preclinical therapy responses, which may inform the next-generation immunotherapy trials

Myeloid lineage switch of Pax5 mutant but not wild-type B cell progenitors by C/EBPα and GATA factors

The developmental potential of hematopoietic progenitors is restricted early on to either the erythromyeloid or lymphoid lineages. The broad developmental potential of Pax5(–/–) pro-B cells is in apparent conflict with such a strict separation, although these progenitors realize the myeloid and erythroid potential with lower efficiency compared to the lymphoid cell fates. Here we demonstrate that ectopic expression of the transcription factors C/EBPα, GATA1, GATA2 and GATA3 strongly promoted in vitro macrophage differentiation and myeloid colony formation of Pax5(–/–) pro-B cells. GATA2 and GATA3 expression also resulted in efficient engraftment and myeloid development of Pax5(–/–) pro-B cells in vivo. The myeloid transdifferentiation of Pax5(–/–) pro-B cells was accompanied by the rapid activation of myeloid genes and concomitant repression of B-lymphoid genes by C/EBPα and GATA factors. These data identify the Pax5(–/–) pro-B cells as lymphoid progenitors with a latent myeloid potential that can be efficiently activated by myeloid transcription factors. The same regulators were unable to induce a myeloid lineage switch in Pax5(+/+) pro-B cells, indicating that Pax5 dominates over myeloid transcription factors in B-lymphocytes

HGAL-a Germinal Center Specific Protein, Enhances B-Cell Receptor Signaling by Activation of Syk, Leading to Follicular Lymphoproliferation

Abstract Abstract 584 The Human Germinal center Associated Lymphoma (HGAL) gene is exclusively expressed in germinal center (GC) B-lymphocytes and GC-derived lymphomas. In patients with diffuse large B-cell lymphomas (DLBCL), HGAL expression identifies a subgroup of patients with biologically distinct tumors associated with improved survival. Our previous in vitro studies demonstrated that HGAL decreases spontaneous and chemoattractant-induced cell motility by activating the RhoA signaling pathway and by directly interacting and augmenting F-actin and myosin II binding. However, the major function of HGAL in GC lymphocytes remains largely unknown. Based on our previous observation of tyrosine phosphorylation of a modified ITAM motif in the HGAL by Lyn, we hypothesized that HGAL may be involved in B-cell receptor (BCR) signaling. Indeed, following BCR stimulation of two GCB-like lymphoma cell lines (Raji and VAL), we observed marked reduction of Syk, Btk and PLCγ phosphorylation upon knockdown of endogenous HGAL by specific but not control siRNAs. Concordantly, HGAL knockdown in BCR-stimulated Raji cells reduced Ca2+ mobilization and decreased NFAT transcriptional activity as analyzed by a luciferase reporter assay. HGAL expression in the BCR-stimulated HBL1 lymphoma cell line (lacking endogenous HGAL protein) resulted in increased Syk, Btk and PLCγ phosphorylation. Syk plays a major role in coupling BCR activation to downstream effectors. Endogenous HGAL was detected in immunoprecipitates of endogenous Syk and vice versa. Nanoscope microscopy studies confirmed co-localization of HGAL and Syk proteins in cell membranes, which was enhanced following BCR stimulation. In BCR-stimulated cells, Syk kinase activity was markedly increased following addition of HGAL protein as measured by an in vitro Syk kinase activity assay. To comprehensively examine HGAL effects on immune system and BCR signaling, we generated a transgenic mouse model in which HGAL is expressed under the control of the mouse Ly-6E.1 promoter in Sca1+ hematopoietic stem cells and progenitors of C57BL/6 × CBA mice. The Sca1-HGAL transgenic mice showed normal embryonic and post natal development, and at 8 weeks of age demonstrated normal lymphoid development without any significant changes in the major hematopoietic compartments (bone marrow (BM), spleen, thymus and peripheral lymph nodes) and in peripheral blood. They also exhibited normal GC development in response to a T-cell dependent antigen immunization. In contrast, at 12 months of age the Sca1-HGAL mice developed a decrease in BM immature B-cells at the expense of recirculating B-cells (B220+IgDhi) compared to the age-matched normal littermates, suggesting a defect in B-cell lymphopoiesis. All the Sca1-HGAL transgenic mice became ill from approximately 12 months of age and all died between 12 to 22 months of age with statistically shorter survival as compared to the wild type controls. Analysis of these animals showed massive splenomegaly with marked white pulp hyperplasia and presence of multiple, frequently contiguous nodules predominantly composed of polyclonal follicular (B220+CD21intCD23hi) B lymphocytes. Extra-lymphatic infiltration by similar B lymphocytes was observed in the liver, lungs and kidneys of Sca1-HGAL mice with advanced disease. IgG isotype titers in these animals tended to be higher than in the wild-type controls, reaching a statistically significant difference for the IgG1 isotype. Follicular hyperplasia in the Sca1-HGAL transgenic mice is likely attributable to increased RhoA activation and enhanced BCR signalling manifested by increased Syk phosphorylation, Ca2+ mobilization and in vitro B cell proliferation following BCR stimulation, in agreement with similar data observed in human DLBCL cell lines expressing HGAL. Gene expression profiling of lymphoid tissues confirmed significantly enhanced BCR signalling and RhoA pathway activation in Sca1-HGAL transgenic mice, corresponding to similar pathway activation in human lymphoma cell lines over-expressing HGAL. Overall, our findings demonstrate that HGAL, specifically expressed in GC B cells, enhances responsiveness to antigens by stimulating Syk kinase activity that without appropriate regulation may lead to lymphoproliferation. Further studies are needed to examine the role of HGAL in the pathogenesis of GC-derived lymphomas. Disclosures: No relevant conflicts of interest to declare

Infection Exposure Is a Causal Factor in B-cell Precursor Acute Lymphoblastic Leukemia as a Result of Pax5-Inherited Susceptibility

Earlier in the past century, infections were regarded as the most likely cause of childhood B-cell precursor acute lymphoblastic leukemia (pB-ALL). However, there is a lack of relevant biologic evidence supporting this hypothesis. We present in vivo genetic evidence mechanistically connecting inherited susceptibility to pB-ALL and postnatal infections by showing that pB-ALL was initiated in Pax5 heterozygous mice only when they were exposed to common pathogens. Strikingly, these murine pB-ALLs closely resemble the human disease. Tumor exome sequencing revealed activating somatic, nonsynonymous mutations of Jak3 as a second hit. Transplantation experiments and deep sequencing suggest that inactivating mutations in Pax5 promote leukemogenesis by creating an aberrant progenitor compartment that is susceptible to malignant transformation through accumulation of secondary Jak3 mutations. Thus, treatment of Pax5(+/-) leukemic cells with specific JAK1/3 inhibitors resulted in increased apoptosis. These results uncover the causal role of infection in pB-ALL development

Homeobox NKX2-3 promotes marginal-zone lymphomagenesis by activating B-cell receptor signalling and shaping lymphocyte dynamics

NKX2 homeobox family proteins have a role in cancer development. Here we show that NKX2-3 is overexpressed in tumour cells from a subset of patients with marginal-zone lymphomas, but not with other B-cell malignancies. While Nkx2-3-deficient mice exhibit the absence of marginal-zone B cells, transgenic mice with expression of NKX2-3 in B cells show marginal-zone expansion that leads to the development of tumours, faithfully recapitulating the principal clinical and biological features of human marginal-zone lymphomas. NKX2-3 induces B-cell receptor signalling by phosphorylating Lyn/Syk kinases, which in turn activate multiple integrins (LFA-1, VLA-4), adhesion molecules (ICAM-1, MadCAM-1) and the chemokine receptor CXCR4. These molecules enhance migration, polarization and homing of B cells to splenic and extranodal tissues, eventually driving malignant transformation through triggering NF-κB and PI3K-AKT pathways. This study implicates oncogenic NKX2-3 in lymphomagenesis, and provides a valid experimental mouse model for studying the biology and therapy of human marginal-zone B-cell lymphomas