Abstract: Acute myeloid leukemia (AML) has a poor prognosis despite intensive therapy. Novel therapies directed at molecular drivers of AML are needed. Ongoing clinical trials with inhibitors of the Menin and Mixed Lineage Leukemia 1 (KMT2A/MLL1) protein-protein interaction for patients with MLL1-rearranged (MLL1-r) and Nucleophosmin mutant (NPM1c) AML have promising early results. We have identified Ikaros degradation as a synergistic therapeutic target with Menin-MLL1 inhibition in MLL1-r AML and identified the novel cereblon E3 ligase modulator (CELMoD) CC-92480 as an efficacious compound in vitro and in vivo in MLL1-r and NPM1c AML models. To find genetic targets that synergize with Menin-MLL1 inhibition, we performed a genome-scale CRISPR-Cas9 functional genetic screen in the Molm-13 (MLL1-AF9) AML cell line. Using the MAGeCKFLUTE pipeline, comparison of 14 days of treatment with VTP-50469 (Menin-MLL1 inhibitor) versus DMSO identified IKZF1 deletion as one of the most negatively selected genes when treated with VTP-50469 (IKZF1 codes for the hematopoietic transcription factor Ikaros). To validate this finding, CRISPR-Cas9-based competition assays were performed in Cas9-expressing MLL1-r and NPM1c AML cell lines by monitoring of sgRNA-RFP expression over time via flow cytometry. IKZF1 was a dependency in all four MLL1-r cell lines and both NPM1c cell lines evaluated. The sgRNAs targeting IKZF1 depleted faster when cells were treated with VTP-50469. To evaluate the effect of Ikaros protein degradation in these AML cell lines, we assessed proliferation upon treatment with three Ikaros-degrading compounds: the Immunomodulatory Imide Drug (IMiD) lenalidomide and the CELMoDs CC-220 and CC-92480. CELMoDs are a derivative class of IMiDs with much more potent Ikaros degradation (lenalidomide & lt; CC-220 & lt; CC-92480) and Phase I activity in multiple myeloma patients refractory to prior IMiD therapy. Cells were plated with 5 different doses per drug. Every 3 days cells were split, drug was replenished, and live cell counts were obtained using viability staining and flow cytometry. Evaluation of the absolute IC 50 in 7 MLL1-r or NPM1c AML cell lines at day 9 showed that 6 cell lines were sensitive to CC-92480 (absolute IC 50 range: & lt;0.1-to-3.6 nM), 5 were sensitive to CC-220 (absolute IC 50 range: 15.7-to-215.3 nM), and only two were sensitive to lenalidomide (absolute IC 50 of 267.0 nM and 555.3 nM). These data suggest that greater Ikaros degradation leads to greater efficacy in vitro. Lenalidomide, CC-220, and CC-92480 all synergized with VTP-50469 to inhibit proliferation. Apoptosis assays were performed in the MLL1-r AML cell lines Molm-13 and MV4;11 using annexin V staining following 6 days of treatment with DMSO, lenalidomide, CC-220, and CC-92480 with and without VTP-50469. For both cell lines, 5 µM lenalidomide, 1 µM CC-220, and 3 nM CC-92480 increased apoptosis less than two-fold as single agents while VTP-50469 did so only 2.5-fold and 3.7-fold compared to DMSO control. Strikingly, the combination of each of these IMiD or CELMoD doses with VTP-50469 induced apoptotic markers at least 7.5-fold compared to DMSO control. This cooperativity between Ikaros protein degradation and Menin-MLL1 inhibition could be explained by their combined targeting of a HOXA/MEIS1 transcriptional program as Ikaros degradation in MLL1-r AML cell lines perturbs expression of HOXA9 target genes without altering the expression of HOXA9 itself. We next tested CC-92480 in four patient derived xenograft (PDX) models of AML: one NPM1c and three MLL1-r models. In these experiments, NSG-S mice were engrafted with PDX samples ( & gt;7 mice per treatment cohort). Following detection of leukemia in peripheral blood, mice were randomized to receive vehicle control, lenalidomide 50 mg/kg (in two of the four PDXs), or CC-92480 10 mg/kg for 28-55 days and survival benefit was assessed. In these models, CC-92480 increased median survival compared to DMSO by 28.3% (p & lt; 0.05), 66% (p & lt;0.0005), 128% (p & lt; 0.0005), and 133% (p & lt; 0.0001). In two of these experiments CC-92480 was also compared to lenalidomide and increased survival by 23.4% (p & lt; 0.0005) and 28.1% (p & lt; 0.05). In summary, the preclinical activity of CC-92480 in MLL1-r and NPM1c AML models, particularly in combination with Menin-MLL1 inhibition, supports translation of this compound or a similarly potent, Ikaros-degrading CELMoD into clinical trials for these molecular subtypes of AML. Disclosures Aubrey: Walter and Eliza Hall Institute of Medical Research: Patents & Royalties: Receives proceeds from Royalties and Milestone payments related to the BCL2-inhibitor, ABT-199/venetoclax. McGeehan: Syndax Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Fischer: Neomorph Inc.: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; C4 Therapeutics: Current equity holder in publicly-traded company; EcoR1 Capital: Consultancy; Sanofi: Consultancy; Astellas: Consultancy; Deerfield: Consultancy; RA Capital: Consultancy; Novartis: Research Funding; Astellas: Research Funding; Deerfield: Research Funding; Ajax: Research Funding; Jengu Therapeutics: Current holder of individual stocks in a privately-held company; Civetta Therapeutics: Current holder of individual stocks in a privately-held company. Armstrong: AstraZeneca: Research Funding; Syndax: Research Funding; Novartis: Research Funding; Janssen: Research Funding; Mana Therapeutics: Consultancy; Accent Therapeutics: Consultancy; OxStem Oncology: Consultancy; C4 Therapeutics: Consultancy; Cyteir Therapeutics: Consultancy; Vitae/Allergan Pharma: Consultancy; Imago Biosciences: Consultancy; Neomorph Inc: Consultancy, Current holder of individual stocks in a privately-held company.

Abstract: Abstract 162 Background: The coinhibitory receptor programmed death (PD)-1 is markedly increased on peripheral blood (PB) and intratumoral T cells in follicular lymphoma (FL) and is associated with impaired T-cell function. CT-011 (pidilizumab), a humanized anti PD-1 monoclonal antibody, blocks the PD-1/PD-ligand pathway and enhances the function of antitumor T and NK cells. We conducted a phase II trial to determine the safety and efficacy of CT-011 and rituximab in patients (pts) with relapsed FL and the clinical results are reported separately. Here, we evaluated the effects of CT-011 on immune cells both in the PB and tumor microenvironment. We also determined predictors of clinical outcome based on PB immune cell subsets and molecular features of tumor-infiltrating immune cells at baseline. Methods: CT-011 was given every 4 wks × 4 and rituximab weekly × 4 starting on day 17 after the first infusion of CT-011. Pts with response or stable disease received 8 additional optional infusions of CT-011 every 4 wks. PB and core needle biopsies from involved lymph nodes were collected prior to and on day 14 after the first infusion of CT-011. PB mononuclear cells (PBMC) were analyzed by multiparametric flow cytometry to determine various immune cell subsets. Whole genome gene expression profiling (GEP) was performed on core needle biopsies. Results: Of 29 pts eligible for efficacy analysis, 19 had an objective response for an ORR of 66%. CR was observed in 15 (52%) and PR in 4 (14%). After a median follow up of 14 mo, median PFS was 21.1 mo, and was not reached for the responders. We observed a significant increase in the absolute number of PB immune cells in day 14 samples compared with baseline including total lymphocyte count (p 〈 0.01), CD3+ T cells (p=0.01), CD4+ T cells (p 〈 0.01), and CD4+ naive (p=0.01), effector memory (p=0.02), and central memory T cells (p 〈 0.05). We also observed increase in the expression of the activating receptor NKG2D on NK cells (p=0.01). In contrast, CD8+ terminally differentiated T cells were decreased (p=0.02). Comparison of GEP from core needle biopsies obtained pretreatment and day 14 (n=8 pairs) showed up regulation of several genes associated with T cell activation in day 14 samples including CD58, CTLA4, NFATC1, CCR5, PBK, TSLP, and ZBTB32. We analyzed GEP of baseline tumor biopsies from 18 pts to find gene signatures that correlated with clinical outcome, as measured by PFS and/or quantitative change in tumor size (%change). We tested a “PD-1hi_Down” signature of 41 genes previously reported by us (Chu et al, Blood, Nov 2011; 118:2648) to be less expressed in CD4+ T cells strongly positive for PD-1, likely to be follicular helper T cells (Tfh, PD-1hi), than in CD4+ T cells with intermediate or low levels of PD-1 surface staining, likely to be exhausted effector T cells (Teffs, PD-1int) or activated effector or naïve T cells (PD-1lo). The PD-1hi_Down gene signature correlated significantly with PFS by univariate Cox regression and was also significant when examined by gene set enrichment analysis based on ranking all genes by correlation with %change. Low expression of this signature, suggesting more Tfh and fewer PD-1+ Teffs within the tumor, predicted shorter PFS duration and less tumor shrinkage. Combined with our in vitro findings that anti-PD-1 Ab enhances the function of Tfh and PD-1+ Teffs but has no effect on PD-1lo T cells in FL, these results suggest that CT-011 therapy enhances the respective pro- and anti-tumor effects of one or both of these cell types. In support of our conclusion that the effect of the PD-1hi_Down signature on outcome depends on CT-011 therapy, we found that this signature did not correlate with overall survival in an external dataset of FL pts treated largely with chemotherapy alone (Dave et al., NEJM 2004; 351: 2109). Conclusions: Administration of CT-011 (pidilizumab) was associated with increase in the numbers of naïve, effector memory, and central memory CD4+ T cells and resulted in activation of T and NK cells in the PB and the tumor microenvironment in FL. A high expression of PD-1hi_Down gene signature, consistent with relatively increased numbers of antitumor Teffs compared with protumor Tfh was predictive of good response and improved PFS suggesting that CT-011 restores function of exhausted Teffs. These results provide insight into the mechanism of action of CT-011 and offer a predictive biomarker for selection of pts for future clinical trials with this class of agents in FL. Disclosures: Rotem-Yehudar: CureTech Ltd: Employment, Research Funding. Neelapu:Cure Tech, Ltd.: Research Funding.

Abstract: Abstract 2766 Background: Lenalidomide plus rituximab therapy is a highly effective and well-tolerated therapy in patients (pts) with follicular lymphoma (FL). In a Phase II trial, this combination induced a complete remission rate of 87% in pts with advanced stage untreated FL (Fowler et al, Ann Oncol, 2011; 22; suppl 4:137). A randomized Phase III trial was recently initiated to compare this combination with current standard of care therapies in pts with FL. Although lenalidomide is known to be an immunomodulatory drug with effects on a variety of immune cells in vitro, its effects have not been well studied in vivo in humans. Understanding the in vivo effects of lenalidomide could lead to novel combination strategies to enhance the efficacy and improve clinical outcome in FL and other malignancies. Methods: Pts received lenalidomide 20 mg/day on days 1–21 of each 28-day cycle and rituximab was given at 375 mg/m2on day 1 of each cycle. Peripheral blood mononuclear cells (PBMC) were phenotyped by multiparametric flow cytometry at baseline, on cycle 2 day 15 (C2D15), and at the end of cycle 6. In addition, peripheral blood (PB) samples were collected in PAXgene Blood RNA tubes at baseline and on C2D15 for whole genome gene expression profiling (GEP). Results: Immunophenotyping of baseline and end of cycle 6 PBMC (n=17) showed that the percentages and absolute numbers of CD3+, CD4+, CD8+, TCRgd, and Foxp3+ regulatory T cells; and NK, NKT, and myeloid dendritic cells were not significantly different between the two time points. However, a significant increase in CD4+CD45RO+ (p 〈 0.01) and CD8+CD45RO+ (p=0.04) memory T cells was observed post-therapy. Further characterization of CD4+ T cells showed a significant increase in central memory T cells (p 〈 0.001) and a decrease in naïve (p 〈 0.01) and terminally differentiated (p 〈 0.01) T cells, but no change in effector memory T cells. The increase in CD8+ central memory T cells was marginally significant (p=0.06). Plasmacytoid dendritic cells (PDC) were also significantly increased (p=0.02). In contrast, no such changes in T cell subsets or PDC were observed in FL pts (n=9) treated with 6 cycles of R-CHOP chemotherapy that received equal number of rituximab doses and analyzed at similar time points (baseline and end of cycle 6). To understand lenalidomide-induced changes on a molecular level, we compared GEP data at C2D15 vs. baseline for 7 pairs of PB samples. The paired significance analysis of microarrays method, based on Student's t test, identified 1,748 differentially expressed genes (DEG; 713 up, 1035 down), without a fold-change threshold, in C2D15 samples vs. baseline. Results were influenced by rituximab-induced depletion of B cells in C2D15 samples, but there were many changes that suggested altered PBMC physiology. Noteworthy up-regulated genes ( 〉 1.5 fold) included genes associated with T and NK cell activation including BATF, CCR2, CD1B, CD2, CD160, CTLA4, CXCR3, ICOS, and LAG3; and CD163 and CD209, phagocytic receptors expressed on monocytes/macrophages. Down-regulated genes ( 〉 1.5 fold) included CXCR5, which mediates B cell migration into follicles; and IL1B and TNFSF13B (BAFF), which are produced by activated macrophages and induce B cell proliferation. Gene set enrichment analysis of all GEP results, and Ingenuity Pathway Analysis of DEGs, indicated up regulation of multiple pathways and processes including ribosomal and mitochondrial components involved in translation and oxidative phosphorylation, CTLA4 signaling in cytotoxic T cells, and differentiation and signaling by ICOS and CD28 in T helper cells. We confirmed up regulation of CTLA4, ICOS, and LAG3 at the protein level in C2D15 PBMC by flow cytometry. Furthermore, treatment of PBMC derived from untreated FL pts with lenalidomide in vitro resulted in up regulation of these molecules in T and/or NK cells consistent with our in vivo results. Conclusions: In FL pts, lenalidomide induced multiple changes in the immune system including increases in PDC and memory T cell subsets, activation of T and NK cells, and down-regulation of certain genes mediating B cell migration and proliferation. These results provide insights into the mechanism of action of lenalidomide and suggest that it can be combined with other immunostimulatory agents such as therapeutic vaccines, adoptive T cell therapy strategies, and immune checkpoint inhibitors to further enhance its efficacy in FL and other malignancies. Disclosures: Fowler: Celgene: Research Funding. Heise:Celgene Corporation: Employment, Equity Ownership. Lacerte:Celgene: Honoraria. Samaniego:Celgene: Research Funding. Neelapu:Celgene Corporation: Research Funding.