R. Eric Davis

BACKGROUND: Flavopiridol, a flavonoid currently in cancer clinical trials, inhibits cyclin-dependent kinases (CDKs) by competitively blocking their ATP-binding pocket. However, the mechanism of action of flavopiridol as an anti-cancer agent has not been fully elucidated. RESULTS: Using DNA microarrays, we found that flavopiridol inhibited gene expression broadly, in contrast to two other CDK inhibitors, roscovitine and 9-nitropaullone. The gene expression profile of flavopiridol closely resembled the profiles of two transcription inhibitors, actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), suggesting that flavopiridol inhibits transcription globally. We were therefore able to use flavopiridol to measure mRNA turnover rates comprehensively and we found that different functional classes of genes had distinct distributions of mRNA turnover rates. In particular, genes encoding apoptosis regulators frequently had very short half-lives, as did several genes encoding key cell-cycle regulators. Strikingly, genes that were transcriptionally inducible were disproportionately represented in the class of genes with rapid mRNA turnover. CONCLUSIONS: The present genomic-scale measurement of mRNA turnover uncovered a regulatory logic that links gene function with mRNA half-life. The observation that transcriptionally inducible genes often have short mRNA half-lives demonstrates that cells have a coordinated strategy to rapidly modulate the mRNA levels of these genes. In addition, the present results suggest that flavopiridol may be more effective against types of cancer that are highly dependent on genes with unstable mRNAs.

Background: Follicular lymphoma (FL) tumors are infiltrated with antitumor T cells, however, their function is impaired by immune checkpoints such as PD-1/PD-ligand pathway. Blocking PD-1 enhances the function of antitumor T cells in FL. In addition, blocking PD-1 on NK cells has been shown to enhance the ADCC effect of NK cells. We reasoned that the combination of pembrolizumab, an anti-PD-1 antibody, and rituximab (R), an anti-CD20 antibody that induces ADCC, is likely to be synergistic through activation of both the innate and adaptive immune systems and result in enhanced clinical activity in FL. Methods: We evaluated pembrolizumab and R in an open-label, non-randomized, single institution, phase II trial (N = 30). Key inclusion criteria included adult (age ≥ 18 years), FL grade 1-3a, ECOG 0-1, in relapse after ≥1 prior therapy and R sensitive disease, defined as a complete (CR) or partial response lasting at least 6 months after most recent R-containing therapy. Patients received R (375 mg/m2 IV) on days 1, 8, 15, and 22 of cycle 1 and pembrolizumab (200 mg IV) q 3 weeks for up to 16 cycles starting on day 2 of cycle 1. Primary endpoint was overall response rate (ORR). Results: 27 patients have initiated therapy, median age 65 (range 42-79), 52% male, 76% had intermediate or high risk FLIPI, 56% met GELF criteria. Median prior therapy =1 (range 1-4). Adverse events (AE) regardless of causality were mild, most grade 1-2. Grade 3 AE's included nausea (N = 2), infusion reaction (N = 2), aseptic meningitis (N = 1), pneumonia (N = 1). Immune-related AEs included grade 2 diarrhea (N = 2), grade 2 pneumonitis (N = 1), grade 2 skin rash (N = 1). At the pre-planned interim analysis (N = 15), ORR was 80%, CR rate was 60%. With a median follow up of 7 months (range 0.5-17), median DOR, PFS, and OS has not been reached. PD-L1 expression was tested in 8 baseline tumor samples using PD-L1 22C3 IHC pharmDx and was detected in histiocytes in all 8 tumors, present in only 1-8% of tumor cells in 5 tumors. Additional biomarker analyses are ongoing. Conclusions: The combination of pembrolizumab and R is well tolerated in relapsed FL and is associated with high overall and CR rate. These interim results warrant further investigation of this combination in FL. Keywords: follicular lymphoma (FL); monoclonal antibodies (MoAb); rituximab.

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.

Background: Diffuse large B-cell lymphoma (DLBCL) is categorized by the cell of origin (COO) classification system into germinal center (GCB) and non-GCB subtypes. Both the immunomodulatory agent lenalidomide (L) and BTK inhibitor ibrutinib (I) have shown promising activity in the non-GCB DLBCL subtype as a single agent and in combination with chemotherapy. Preclinical studies demonstrate L + I results in synthetic lethality in non-GCB DLBCL via interferon signaling (Yang et al; Cancer Cell, 2012), but no trials have evaluated the efficacy in newly diagnosed patients. We present the first “window of opportunity” trial to use targeted therapy (rituximab, R + L + I) prior to chemotherapy in newly diagnosed DLBCL patients. Methods: In this investigator-initiated phase II trial, patients receive standard R q 21 days (d), L 25 mg po d1-10, I 560 mg po d1-21, and after two 21d RLI cycles or with progression start standard chemotherapy (initially dose adjusted EPOCH q 21d for 6 cycles, but the randomized 50303 study results prompted an amendment to allow either EPOCH or CHOP). L + I dosing may be reduced if significant toxicity criteria are met. Key eligibility criteria include histologically confirmed, treatment-naïve non-GCB DLBCL, age ≥ 18 years, and measurable disease. COO will be determined via immunohistochemistry (IHC) for eligibility and NanoString for confirmation. Patients are restaged with PET/CT (Lugano criteria, Cheson et al, JCO 2014). The primary objectives are to determine the overall response rate (ORR) after 2 cycles of RLI and complete response (CR) rate after 6 cycles of RLI + chemotherapy. Secondary objectives include safety and survival outcomes. Exploratory objectives include evaluation of baseline and therapy induced changes in gene and protein expression, mutations, and immune cell subsets in comparison with clinical outcomes. The trial will accrue 60 patients with Bayesian futility and toxicity monitoring rules. Results: At data cutoff of 3/15/17, 13 patients have enrolled, 1 withdrew consent after 1 cycle, all are available for toxicity and 11 for efficacy. The median age is 65 years, 75% are female, and the median IPI is 2 with 42% having an IPI of 3-5. All patients are non-GCB via IHC testing. Toxicities are noted for no significant rash in any patients and one case of invasive asperigillosis of the brain on a patient treated with high-dose steroids. All patients have efficacy reassessment after RLI window with an ORR of 82% and CR rate of 42%. The 6 patients who have completed all therapy have all achieved a CR. Conclusions: The combination of RLI has shown impressive efficacy both alone and with chemotherapy in this ongoing study in newly diagnosed non-GCB DLBCL. One patient had an invasive fungal infection, prompting a protocol amendment to prohibit steroid use during the window portion, and no further events have occurred. Additional results will be presented at the meeting. Keywords: diffuse large B-cell lymphoma (DLBCL); ibrutinib; lenalidomide