Macrophages (MPs) are heterogeneous, multifunctional, myeloid-derived leukocytes that are part of the innate immune system, playing wide-ranging critical roles in basic biological activities, including maintenance of tissue homeostasis involving clearance of microbial pathogens. Tumor-associated MPs (TAMs) are MPs with defined specific M2 phenotypes now known to play central roles in the pathophysiology of a wide spectrum of malignant neoplasms. Also, TAMs are often intrinsic cellular components of the essential tumor microenvironment (TME). In concert with lymphoid-lineage B and T cells at various developmental stages, TAMs can mediate enhanced tumor progression, often leading to poor clinical prognosis, at least partly through secretion of chemokines, cytokines, and various active proteases shown to stimulate tumor growth, angiogenesis, metastasis, and immunosuppression. Researchers recently showed that TAMs express certain key checkpoint-associated proteins [e.g., programmed cell death protein 1 (PD-1), programmed cell death-ligand 1 (PD-L1)] that appear to be involved in T-cell activation and that these proteins are targets of other specific checkpoint-blocking immunotherapies (anti-PD-1/PD-L1) currently part of new therapeutic paradigms for chemotherapy-resistant neoplasms. Although much is known about the wide spectrum and flexibility of MPs under many normal and neoplastic conditions, relatively little is known about the increasingly important interactions between MPs and B-lymphoid cells, particularly in the TME in patients with aggressive B-cell non-Hodgkin lymphoma (NHL-B). Normal and neoplastic lymphoid and myeloid cell/MP lineages appear to share many primitive cellular characteristics as well as transcriptional factor interactions in human and animal ontogenic studies. Such cells are capable of ectopic transcription factor-induced lineage reprogramming or transdifferentiation from early myeloid/monocytic lineages to later induce B-cell lymphomagenesis in experimental in vivo murine systems. Close cellular interactions between endogenous clonal neoplastic B cells and related aberrant myeloid precursor cells/MPs appear to be important interactive components of aggressive NHL-B that we discuss herein in the larger context of the putative role of B-cell/MP cellular lineage interactions involved in NHL-B pathophysiology during ensuing lymphoma development.

B-cell lymphoma-2 (BCL-2), an antiapoptotic protein often dysregulated in B-cell lymphomas, promotes cell survival and provides protection from stress. A recent phase I first-in-human study of the BCL-2 inhibitor venetoclax in non-Hodgkin lymphoma showed an overall response rate of 44%. These promising clinical results prompted our examination of the biological effects and mechanism of action underlying venetoclax activity in aggressive B-cell lymphoma, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). MCL and DLBCL cell lines, primary patient samples, and patient-derived xenograft (PDX) models were utilized to examine venetoclax efficacy. Furthermore, the mechanisms underlying venetoclax response and the development of venetoclax resistance were evaluated using proteomics analysis and Western blotting. Potential biomarkers linked to venetoclax activity and targeted combination therapies that can augment venetoclax response were identified. We demonstrate that DLBCL and MCL cell lines, primary patient samples, and PDX mouse models expressing high BCL-2 levels are extremely sensitive to venetoclax treatment. Proteomics studies showed that venetoclax substantially alters the expression levels and phosphorylation status of key proteins involved in cellular processes, including the DNA damage response, cell metabolism, cell growth/survival, and apoptosis. Short- and long-term exposure to venetoclax inhibited PTEN expression, leading to enhanced AKT pathway activation and concomitant susceptibility to PI3K/AKT inhibition. Intrinsic venetoclax-resistant cells possess high AKT activation and are highly sensitive to PI3K/AKT inhibition. These findings demonstrate the on-target effect of venetoclax and offer potential mechanisms to overcome acquired and intrinsic venetoclax resistance through PI3K/AKT inhibition. .

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Mantle Cell Lymphoma (MCL) is a relatively uncommon, aggressive form of B-cell non-Hodgkin’s lymphoma (NHL), defining approximately 5-8% of adult NHL in the United States. Although individual survival times have increased in recent years, prognosis remains among the poorest of NHL, with a median survival of 5 to 7 years. Despite an initial response to chemotherapy, MCL cells remain residually in the marrow and digestive tract, making MCL prone to recur leading to short survival prognosis. The characteristic re-emergence of MCL is likely related to drug resistance generated from the tumor microenvironment (TME). In particular, stromal cells found in bone marrow and secondary lymphoid sites promote the movement and proliferation and possibly drug resistance from cross-talk between stromal and lymphoma cells. While studies show a correlation between stromal cells and tumor survival in various forms of NHL, the mechanisms are poorly understood and its role in MCL has not extensively explored. Based on our experimental studies on an extensive spectrum of representative patient samples, we have shown that in vitro culturing of primary leukemic phase MCL cells (15 cases) usually leads to the spontaneous formation of activated CD68+ monocytic macrophages and stromal cells. The MCL tumor cells tended to form rosettes, clustering around and adhering to these cultured lymphoma-associated macrophages (LAM). The LAM formed in culture coexisted with rosetted MCL tumor cells for up to 4 months. During this period, a small subset of MCL cells showed limited mitotic figures, suggesting that these cells were proliferating and possibly becoming incipient immortalized MCL cells. When these cells were weaned from the adherent LAM, they began to expand autonomously and proliferate as an autonomous cell line. Using this methodology, we have developed 3 MCL cell lines (PF-1, PF-2, and PF-3) from 15 leukemic phase MCL cases. PF-1 MCL cell line represents classic typical MCL cells, while PF-2 and PF-3 MCL cell lines represent the in situ indolent MCL cells that are CD200 positive. When these cells were xeno-transplanted into SCID mice, lymphoma cell uptakes were disseminated throughout the lymphatic system, including the spleen, lymph nodes, and GI tract, representing excellent in vivo models for MCL. Next, we delineate the biologic functions of LAM in the MCL microenvironment milieu. Our data showed that when we initially removed the accessory cells (monocytes, etc.) from the MCL primary culture, no LAM were formed and the purified MCL cells died out in <2-3 weeks in culture, suggesting that the LAM developed in culture are biologically and pathologically functional in providing growth/survival signals to the tumor cells. We then examined several therapeutic agents that have shown efficacy against relapsed-MCL in the clinic, including the proteasome inhibitor carfilzomib (CFZ) and the BTK inhibitor ibrutintib (IB), on targeting LAM. Our results showed that both CFZ and IB, at very low drug concentrations could affectively eliminate LAM, followed by spontaneous apoptosis of the MCL cells. We have also developed a unique mesenchymal stromal cell line (PF-MSC), derived from a leukemic phase MCL patient. These cells grow in culture as spindle-shaped morphologies and can enhance the growth of MCL cells as well as protect MCL cells from chemotherapy. However, PF-MSC cells did not protect MCL cells from CFZ or IB therapies. Our findings clearly indicate that MCL TME is a critical mediator for growth/survival and chemoresistance mechanisms in the pathophysiology of MCL. Therapeutically targeting these MCL-associated macrophages/stromal cells with CFZ and/or IB should lead to better therapeutic strategies for refractory MCL patients. Disclosures Pham: Onyx/Amgen: Research Funding.