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© 2019 American Association for Cancer Research. The extraordinary activity of high-dose cyclophosphamide against some high-grade lymphomas was described nearly 60 years ago. Here we address mechanisms that mediate cyclophosphamide activity in bona fide human double-hit lymphoma. We show that antibody resistance within the bone marrow (BM) is not present upon early engraftment but develops during lymphoma progression. This resistance required a high tumor:macrophage ratio, was recapitulated in spleen by partial macrophage depletion, and was overcome by multiple, high-dose alkylating agents. Cyclophosphamide induced endoplasmic reticulum (ER) stress in BM-resident lymphoma cells in vivo that resulted in ATF4-mediated paracrine secretion of VEGFA, massive macrophage infiltration, and clearance of alemtuzumab-opsonized cells. BM macrophages isolated after cyclophosphamide treatment had increased phagocytic capacity that was reversed by VEGFA blockade or SYK inhibition. Single-cell RNA sequencing of these macrophages identified a “super-phagocytic” subset that expressed CD36/FCGR4. Together, these findings define a novel mechanism through which high-dose alkylating agents promote macrophage-dependent lymphoma clearance. SIGNIFICANCE: mAbs are effective against only a small subset of cancers. Herein, we recapitulate compartment-specific antibody resistance and define an ER stress–dependent mechanism induced by high-dose alkylating agents that promotes phagocytosis of opsonized tumor cells. This approach induces synergistic effects with mAbs and merits testing across additional tumor types.

Despite decades of clinical use, mechanisms of glucocorticoid resistance are poorly understood. We treated primary murine T lineage acute lymphoblastic leukemias (T-ALLs) with the glucocorticoid dexamethasone (DEX) alone and in combination with the pan-PI3 kinase inhibitor GDC-0941 and observed a robust response to DEX that was modestly enhanced by GDC-0941. Continuous in vivo treatment invariably resulted in outgrowth of drug-resistant clones, ~30% of which showed markedly reduced glucocorticoid receptor (GR) protein expression. A similar proportion of relapsed human T-ALLs also exhibited low GR protein levels. De novo or preexisting mutations in the gene encoding GR (Nr3c1) occurred in relapsed clones derived from multiple independent parental leukemias. CRISPR/Cas9 gene editing confirmed that loss of GR expression confers DEX resistance. Exposing drug-sensitive T-ALLs to DEX in vivo altered transcript levels of multiple genes, and this response was attenuated in relapsed T-ALLs. These data implicate reduced GR protein expression as a frequent cause of glucocorticoid resistance in T-ALL.

The extraordinary activity of high-dose cyclophosphamide against some high-grade lymphomas was described nearly 60 years ago. Here we address mechanisms that mediate cyclophosphamide activity in bona fide human double-hit lymphoma. We show that antibody resistance within the bone marrow (BM) is not present upon early engraftment but develops during lymphoma progression. This resistance required a high tumor:macrophage ratio, was recapitulated in spleen by partial macrophage depletion, and was overcome by multiple, high-dose alkylating agents. Cyclophosphamide induced endoplasmic reticulum (ER) stress in BM-resident lymphoma cells in vivo that resulted in ATF4-mediated paracrine secretion of VEGFA, massive macrophage infiltration, and clearance of alemtuzumab-opsonized cells. BM macrophages isolated after cyclophosphamide treatment had increased phagocytic capacity that was reversed by VEGFA blockade or SYK inhibition. Single-cell RNA sequencing of these macrophages identified a super-phagocytic subset that expressed CD36/FCGR4. Together, these findings define a novel mechanism through which high-dose alkylating agents promote macrophage-dependent lymphoma clearance. SIGNIFICANCE: mAbs are effective against only a small subset of cancers. Herein, we recapitulate compartment-specific antibody resistance and define an ER stress-dependent mechanism induced by high-dose alkylating agents that promotes phagocytosis of opsonized tumor cells. This approach induces synergistic effects with mAbs and merits testing across additional tumor types.

The extraordinary activity of high-dose cyclophosphamide against some high-grade lymphomas was described nearly 60 years ago. Here we address mechanisms that mediate cyclophosphamide activity in bona fide human double-hit lymphoma. We show that antibody resistance within the bone marrow (BM) is not present upon early engraftment but develops during lymphoma progression. This resistance required a high tumor:macrophage ratio, was recapitulated in spleen by partial macrophage depletion, and was overcome by multiple, high-dose alkylating agents. Cyclophosphamide induced endoplasmic reticulum (ER) stress in BM-resident lymphoma cells in vivo that resulted in ATF4-mediated paracrine secretion of VEGFA, massive macrophage infiltration, and clearance of alemtuzumab-opsonized cells. BM macrophages isolated after cyclophosphamide treatment had increased phagocytic capacity that was reversed by VEGFA blockade or SYK inhibition. Single-cell RNA sequencing of these macrophages identified a super-phagocytic subset that expressed CD36/FCGR4. Together, these findings define a novel mechanism through which high-dose alkylating agents promote macrophage-dependent lymphoma clearance. SIGNIFICANCE: mAbs are effective against only a small subset of cancers. Herein, we recapitulate compartment-specific antibody resistance and define an ER stress-dependent mechanism induced by high-dose alkylating agents that promotes phagocytosis of opsonized tumor cells. This approach induces synergistic effects with mAbs and merits testing across additional tumor types.