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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. See related commentary by Duval and De Palma, p. 834 . This article is highlighted in the In This Issue feature, p. 813 ., (©2019 American Association for Cancer Research.)

Focal amplification of chromosome 1q23.3 in patients with advanced primary or relapsed urothelial carcinomas is associated with poor survival. We interrogated chromosome 1q23.3 and the nearby focal amplicon 1q21.3, as both are associated with increased lymph node disease in patients with urothelial carcinoma. Specifically, we assessed whether the oncogene MCL1 that resides in 1q21.3 and the genes that reside in the 1q23.3 amplicon were required for the proliferation or survival of urothelial carcinoma. We observed that suppressing MCL1 or the death effector domain-containing protein (DEDD) in the cells that harbor amplifications of 1q21.3 or 1q23.3, respectively, inhibited cell proliferation. We also found that overexpression of MCL1 or DEDD increased anchorage independence growth in vitro and increased experimental metastasis in vivo in the nonamplified urothelial carcinoma cell line, RT112. The expression of MCL1 confers resistance to a range of apoptosis inducers, while the expression of DEDD led to resistance to TNFα-induced apoptosis. These observations identify MCL1 and DEDD as genes that contribute to aggressive urothelial carcinoma. IMPLICATIONS: These studies identify MCL1 and DEDD as genes that contribute to aggressive urothelial carcinomas., (©2019 American Association for Cancer Research.)

Inactivation of the retinoblastoma gene ( RB1 ) product, pRB, is common in many human cancers. Targeting downstream effectors of pRB that are central to tumorigenesis is a promising strategy to block the growth of tumors harboring loss-of-function RB1 mutations. One such effector is retinoblastoma-binding protein 2 (RBP2, also called JARID1A or KDM5A), which encodes an H3K4 demethylase. Binding of pRB to RBP2 has been linked to the ability of pRB to promote senescence and differentiation. Importantly, genetic ablation of RBP2 is sufficient to phenocopy pRB's ability to induce these cellular changes in cell culture experiments. Moreover, germline Rbp2 deletion significantly impedes tumorigenesis in Rb1 +/- mice. The value of RBP2 as a therapeutic target in cancer, however, hinges on whether loss of RBP2 could block the growth of established tumors as opposed to simply delaying their onset. Here we show that conditional, systemic ablation of RBP2 in tumor-bearing Rb1 +/- mice is sufficient to slow tumor growth and significantly extend survival without causing obvious toxicity to the host. These findings show that established Rb1 -null tumors require RBP2 for growth and further credential RBP2 as a therapeutic target in human cancers driven by RB1 inactivation., Competing Interests: The authors declare no conflict of interest.

Purpose: Although significant progress has been made in the treatment of T-cell acute lymphoblastic leukemia (T-ALL), many patients will require additional therapy for relapsed/refractory disease. Cyclin D3 (CCND3) and CDK6 are highly expressed in T-ALL and have been effectively targeted in mutant NOTCH1-driven mouse models of this disease with a CDK4/6 small-molecule inhibitor. Combination therapy, however, will be needed for the successful treatment of human disease. Experimental Design: We performed preclinical drug testing using a panel of T-ALL cell lines first with LEE011, a CDK4/6 inhibitor, and next with the combination of LEE011 with a panel of drugs relevant to T-ALL treatment. We then tested the combination of LEE011 with dexamethasone or everolimus in three orthotopic mouse models and measured on-target drug activity. Results: We first determined that both NOTCH1 -mutant and wild-type T-ALL are highly sensitive to pharmacologic inhibition of CDK4/6 when wild-type RB is expressed. Next, we determined that CDK4/6 inhibitors are antagonistic when used either concurrently or in sequence with many of the drugs used to treat relapsed T-ALL (methotrexate, mercaptopurine, asparaginase, and doxorubicin) but are synergistic with glucocorticoids, an mTOR inhibitor, and gamma secretase inhibitor. The combinations of LEE011 with the glucocorticoid dexamethasone or the mTOR inhibitor everolimus were tested in vivo and prolonged survival in three orthotopic mouse models of T-ALL. On-target activity was measured in peripheral blood and tissue of treated mice. Conclusions: We conclude that LEE011 is active in T-ALL and that combination therapy with corticosteroids and/or mTOR inhibitors warrants further investigation. Clin Cancer Res; 23(4); 1012-24. ©2016 AACR See related commentary by Carroll et al., p. 873 ., (©2016 American Association for Cancer Research.)