Citations

Abstract Background: Synthetic lethality (SL) is characterized by the chemical inhibition of multiple aberrant genes to differentially kill malignant cells. We investigated small-molecule combinations for SL through in vitro and in vivo screening and optimization studies (as in the granted patents in USA and China) and demonstrated the synergistic potential of best-in-class combinations of targeted agents, drug repositioning, immune modulation and low-dose combinations. We hypothesize that BTK and mTOR inhibition coupled with an IMID will target multiple key signaling pathways, improve differential apoptosis and target acquired drug resistance. DTRM-555, an optimized mechanism-based combination, consists of the clinically differentiated BTK inhibitor DTRMWXHS-12 (or DTRM-12), everolimus (EV) and pomalidomide (POM). In xenograft tumor models, DTRM-555 administered orally has demonstrated superior efficacy over monotherapy at very low dose combinations of the three agents (1/18 of DTRM-12, 1/6 of EV, and 1/6 of POM). DTRM-12 monotherapy has been safe and well tolerated in simultaneous phase Ia trials (US and China) with preliminary efficacy across 50 mg through 400 mg once daily (QD). Here we report the US clinical results of the oral combination therapies DTRM-505 (DTRM-12 plus EV) and DTRM-555 (DTRM-12 plus EV plus POM) for the first time (NCT02900716). Methods: The safety and anti-tumor activity of the oral doublet therapy DTRM-505 and the oral triplet therapy DTRM-555 are evaluated using a "3+3" study design. Enrollment was conducted concurrently with a phase Ia study of DTRM-12 monotherapy; with DTRM-505 opened for accrual when monotherapy DTRM-12 cleared the 200-mg dose level. Eligible patients are at least 18 years old, having ECOG performance status less than 2 with relapsed/refractory chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL) or classical Hodgkin lymphoma (cHL). Study treatment is administered daily for 21 consecutive days over a 28-day cycle. The dose-limiting toxicity (DLT) period was defined during the first cycle of therapy, and treatment was continued until disease progression or unacceptable toxicity. Patients underwent tumor evaluations every 2 months and response was recorded using revised response criteria (IWCLL and Cheson 2014). Results: Between 9/2016 and 7/2018, 19 patients have been treated with DTRM-12 monotherapy in the USA (6 pts, 50-300 mg) and China (13 pts, 50-400 mg). Patients have tolerated up to 23+ cycles of DTRM-12 monotherapy. Seven patients, including DLBCL, CLL, MCL, FL and cHL, have been treated with oral doublet therapy DTRM-505 for 2 to 12 cycles. Three patients (2 DLBCL, 1 FL) have been treated with oral triplet therapy DTRM-555, including one patient transitioned from the doublet DTRM-505 under an exploratory arm. No patient has discontinued due to an adverse event (AE) and no DLT has occurred. Recurring AEs are mostly grade 1 or 2 and responses have been seen with DTRM-505 and DTRM-555 across diverse lymphoma histologies (See Tables). All patients evaluable for response demonstrated partial response to DTRM-505 and DTRM-555, with 2/7 (doublet) and 3/3 (triplet) patients remaining on therapy. Conclusion: The maximum tolerated dose has not been reached for DTRM-12 monotherapy and for the current fixed dose doublet and triplet combinations. Toxicities have been mild and repeated cycles of study treatment have been well tolerated without delays of subsequent treatment cycles. Enrollment continues for refractory lymphoma patients to demonstrate the safety and efficacy for the first-in-class oral triplet therapy DTRM-555. Disclosures Huntington: Janssen: Consultancy; Bayer: Consultancy; Celgene: Consultancy. Schuster:Merck: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; OncLive: Honoraria; Dava Oncology: Consultancy, Honoraria; Physician's Education Source, LLC: Honoraria; Nordic Nanovector: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees. He:Zhejiang DTRM Biopharma Co. Ltd.: Other: Founder and CEO; Zhejiang DTRM Biopharma LLC: Other: Founder and CEO. Shen:Zhejiang DTRM Biopharma LLC: Other: Clinical Operations Director. Kennard:AbbVie, Gilead, Verastem: Consultancy. Dwivedy Nasta:Incyte: Research Funding; Takeda/Millenium: Research Funding; Debiopharm: Research Funding; Pharmacyclics: Research Funding; Rafael/WF: Research Funding; Aileron: Research Funding; Roche: Research Funding; Merck: Other: DSMC; Celgene: Membership on an entity's Board of Directors or advisory committees. Landsburg:Takeda: Consultancy; Curis: Consultancy, Research Funding. Porter:Novartis: Other: Advisory board, Patents & Royalties, Research Funding; Genentech: Other: Spouse employment; Kite Pharma: Other: Advisory board. Svoboda:Bristol-Myers Squibb: Consultancy, Research Funding; TG Therapeutics: Research Funding; Kyowa: Consultancy; Regeneron: Research Funding; Merck: Research Funding; Seattle Genetics: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; KITE: Consultancy. Song:Peking University Cancer Hospital (Beijing Cancer Hospital): Employment. Zhu:Beijing Cancer Hospital: Employment. Brander:Pharmacyclics, an AbbVie Company: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; TG Therapeutics: Consultancy, Honoraria, Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; Novartis: Consultancy, Other: DSMB; Genentech: Consultancy, Honoraria, Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; Teva: Consultancy, Honoraria; Acerta: Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; DTRM: Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; BeiGene: Other: Institutional research funding for non investigator initiated clinical trial, Research Funding. Ding:Merck: Research Funding. Mato:Pharmacyclics, an AbbVie Company: Consultancy, Research Funding; AstraZeneca: Consultancy; Regeneron: Research Funding; Celgene: Consultancy; Acerta: Research Funding; TG Therapeutics: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Portola: Research Funding; Johnson & Johnson: Consultancy; Prime Oncology: Honoraria; Medscape: Honoraria.

Abstract Introduction: Given the established role of PD-1 in mediating immune suppression in chronic lymphocytic leukemia (CLL), we tested and reported the efficacy of PD-1 blocking antibody pembrolizumab in relapsed and transformed CLL patients. A selective response of pembrolizumab (~40%) in patients with RT, particularly after prior ibrutinib, was observed (Ding, Blood, 129:3419). Correlative analysis showed PD-1 expression in tumor B-cells of patients with RT and aggressive CLL after progression on ibrutinib. PD-1, an inhibitory receptor expressed on CLL T-cells, inhibits the immune synapse and cytotoxic T cell functions via the interactions with its ligands. However, the expression pattern and role of PD-1 in tumor B-cells is not well defined. In this study we investigated the functional implication of the PD-1 signaling axis in B-cell pathobiology in CLL and RT patients. Methods: 26 CLL-involved lymph node (LN) and 20 RT-involved LN were tested for PD-1 expression by immunohistochemistry (mouse clone NAT105, Abcam). For in vitro study, we checked PD-1 expression in 11 lymphoma cell lines and 1 CLL cell line by both flow cytometry and Western blot (WB) analysis. Effect of PD-1 knockdown using CRISPR/Cas9 system (Addgene) and over-expression of PD-1 using pLEX-lentiviral (Thermoscientific) or pRetro-retroviral (Clonetech) system were evaluated on pro-survival and apoptotic signaling pathways by Western blot analysis. Gene expression signatures in CLL and RT patients were also evaluated by Illumina-based RNA sequencing using FFPE-nodal tissue obtained by clinical biopsy (Tempus Labs; Chicago, IL). Results: The expression of PD-1 was significantly increased in RT-LN compared to CLL-LN. (mean ± SEM in RT vs. CLL, 30.6 ± 4.7 vs. 11.5 ± 2.8, p < 0.001). PD-1 expression was highest in patients with RT where the immediate prior CLL therapy was ibrutinib (Figure 1A). Among all cell lines tested for PD-1 expression, the expression of PD-1 by WB and flow was highest in Mino (mantle cell lymphoma line), followed by moderate expression in Jvm2 (B-PLL line) and Mec1 (CLL line), and very low-level expression in both Jeko-1 (B-NHL line) and lymphoma line 'Karpas299'. CRISPR/Cas9 mediated depletion of PD-1 in Mino cells inhibited constitutively active Akt, p70S6K and mTOR pathway, accompanied by significant downregulation of the anti-apoptotic proteins, Bcl-2, Mcl-1 and XIAP, but P-ERK1/2 was not affected. Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in Jeko-1 and doxycycline regulated inducible retroviral (pRetro-PD-1) mediated overexpression of PD-1 in Karpas299 activated Akt, mTOR and p70S6K pathway. Overexpression of PD-1 in Jeko-1 significantly increased Bcl-2 and Mcl-1 and in Karpas299 increased Bcl-2, Mcl-1 and XIAP expression (Figure 1B). A parallel genetic analysis using RNA sequencing was performed on 5 nodal tissues involved by either RT or progressive CLL after these patients developed clinical progression after prior ibrutinib therapy. In all 5 patients, overexpression of PD-1 was associated with increased expression of Bcl-2 and mTOR regardless of the genetic mutations detected (including TP53, ATM, BTK, NOTCH1, XPO1, SF3B1, TET2 etc). However, in 2 patients who received prior chemoimmunotherapy, similar overexpression of gene signature was not observed by RNA sequencing analysis, alternative pathways including Met or NFkB overexpression was detected. Given these clinical and laboratory findings, we have treated 2 RT patients with a combination of BTK and Bcl-2 inhibitors (ibrutinib and venetoclax, respectively) whose CLL transformed after prior ibrutinib. Significant reduction of tumor burden was observed in both cases with one complete response and one mixed response. Conclusion: An increased expression of PD-1, Akt/mTOR and Bcl-2 gene signature was first observed in RT patients after prior ibrutinib therapy. PD-1 overexpression in the tumor B-cells of RT and progressive CLL patients likely regulate AKT/mtOR to upregulate Bcl-2. Targeting both BTK and Bcl-2 pathways in addition to PD-1 blockade appear to be a promising strategy to treat these aggressive diseases. Disclosures Parikh: Gilead: Honoraria; Janssen: Research Funding; Abbvie: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding. Kenderian:Novartis: Patents & Royalties; Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding. Ansell:Takeda: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Celldex: Research Funding; Merck & Co: Research Funding; Regeneron: Research Funding; LAM Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Research Funding; Pfizer: Research Funding. Kay:Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees. Ding:Merck: Research Funding.

Abstract Peripheral immune dysfunction in B-Chronic Lymphocytic Leukemia (CLL) is well-studied and likely relates to the incidence of serious recurrent infections and second malignancies that plague CLL patients. However, the current paradigms of known immune abnormalities are not able to consistently explain these complications and it is not easy to correct CLL patient immune status. Here, we expand on our preliminary reports that demonstrate bone marrow (BM) hematopoietic dysfunction in early and late stage untreated CLL patients. We found reduced short-term functional capacity of hematopoietic progenitors in BM using colony forming unit assays (Figure 1A-C) and flow cytometry revealed significant reductions in frequencies of hematopoietic stem and progenitor cell (HSPC) populations (exemplified by Lin-CD34+ HSPCs, Figure 1D). We further report that protein levels of the transcriptional regulators HIF-1α, GATA-1, PU.1, and GATA-2 are overexpressed in distinct HSPC subsets from CLL patient BM, providing molecular insight into the basis of HSPC dysfunction. Interestingly, sustained myelopoiesis, evaluated by limiting dilution analysis in long-term culture-initiating cell (LTC-IC) assays maintained for five weeks, revealed no difference between healthy controls and CLL patients. These new data indicate that when HSPCs are removed from the leukemic microenvironment for ample in vitro culture time, they recover the ability to sustain myelopoiesis. To further assess the impact of the CLL microenvironment on HSPC biology, isolated HSPCs (CD34+ BM cells) from healthy controls were exposed in vitro to known leukemic microenvironment constituents. Exposure to TNFα, a cytokine constitutively produced by CLL B cells, resulted in rapid increases in PU.1 and GATA-2 proteins (Figure 2A-D). Similarly, addition of TNFα to the LTC-IC assay resulted in a striking ablation of myelopoiesis, even at the highest input cell concentration. Further, overexpression of PU.1 and GATA-2 were observed in HSPCs following co-culture with CLL B cells, a result that was not recapitulated when cells were exposed to IL-10, another cytokine constitutively produced by CLL B cells. These findings indicate specific components of the leukemic microenvironment are involved in HSPC modulation. Together, these findings expand on our previous observations of BM hematopoietic dysfunction in untreated CLL patients and offer new molecular insights into the contribution of the leukemic microenvironment on immunodeficiency in CLL. Disclosures Ding: Merck: Research Funding. Parikh:Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; Janssen: Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Honoraria; AstraZeneca: Honoraria, Research Funding. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract Background Predictors of outcomes after reduced intensity conditioning (RIC) stem cell transplantation (SCT) in chronic lymphocytic leukemia (CLL) are not well established. The Dana Farber (DF) investigators (Brown et al, leukemia 2013) developed a prognostic scoring model for overall survival (OS) and disease free survival (DFS) that assigned one point to each of comorbidity index, absolute lymphocyte count, LDH and disease status. The goals of the present study were to 1) validate the DF model 2) identify additional predictors of outcomes after SCT. Methods This study is a retrospective review of the Mayo Clinic CLL database and was approved by the Institutional Review Board. All CLL patients that underwent RIC-SCT were included. The prognostic significance of prognostic factors at time of RIC-SCT were analyzed, including the DF model. Survival was estimated and compared using the Kaplan Meier and Log Rank tests. Results From 2006 to 2012, 50 patients with CLL underwent RIC-SCT at the Mayo Clinic. Of these, 33 were male (66%) and the median age was 56 (29-69) years. The median time from diagnosis to RIC-SCT was 4.7 years (0.6-22.9). Indications for RIC-SCT were 17p deletion in 24 (48%) patients and purine nucleoside analogue (PNA) refractory disease in 26 (52%) patients. Transplant related mortality was 6% and non-relapse mortality at 5 years was 14%. Acute graft versus host disease (GVHD) developed in 30 (60%) patients and chronic (c) GVHD was noted in 32 patients (64%). The 5-year relapse rate was 38%. The DF prognostic score stratified three categories for OS (score of 0, 1-2, ≥ 3), but was not associated with DFS in this cohort. We next evaluated the impact of other patient, disease, and transplant characteristics on clinical outcome. Development of cGVHD post-transplant was the dominant predictor of both DFS (HR 0.26, 95% CI=0.10-0.69, P=0.006) and OS (HR 0.04, 95% CI=0.01-0.19, P<0.0001, figure 1). The presence of 17p deletion at time of RIC-SCT was associated with shorter DFS (HR 3.99, 95% CI=1.43-11.18, P=0.008), but did not impact OS. Developing cGVH but not DF score remained an independent predictor of OS on a multi-variate analysis including both factors. Given these findings, we evaluated what pre-treatment patient, disease, and transplant characteristics predicted for subsequent development of cGVHD. Patients with higher scores on DF prognostic model were less likely to develop cGVHD (score 0=66%, 1-2=76%, ≥3=31%; P=0.01). Three CLL characteristics (complete or partial remission at time of RIC-SCT, absence of 17p deletion, and absence of ZAP70 overexpression) were predictors for development of cGVHD while alemtuzumab exposure ≤3 months prior to RIC-SCT decreased likelihood of cGVHD. On multivariate modeling using these four predictors, only ZAP70 overexpression was an independent negative predictor of developing cGVHD (Table 1). Rates of cGVHD post RIC-SCT in ZAP 70 positive and ZAP70 negative patients were 50% and 92%, respectively (p=0.03). Conclusion This study found that while the DF prognostic score is an effective way to predict outcome prior to RIC-SCT, development of cGVHD post-transplant is ultimately the most significant predictor for both OS and DFS after transplant in patients with CLL. Pretransplant characteristics that predict subsequent cGVHD include DF prognostic score and ZAP70 status. This is the first report to identify biologic characteristics of CLL B-cells associated with development of cGVHD post-transplant. Disclosures: No relevant conflicts of interest to declare.

Abstract Clonal hematopoiesis of indeterminate potential (CHIP) is defined as somatic mutations in clonal blood cells of individuals without any other hematological abnormalities and have been identified using whole exome sequencing (WES) from pre-specified genes. CHIP is reportedly associated with aging, reduced overall survival, cardiovascular disease, and hematologic disease, mainly myeloid cancers. Additionally, whole genome sequencing and WES studies in CLL cases have identified putative CLL driver genes with recurrent somatic variants or copy number alterations. Here, we investigate the associations of CHIP and mutations in CLL somatic driver genes with risk of CLL and MBL, a precursor to CLL. Using WES, 571 individuals from 132 CLL families were sequenced from two different sites, Mayo Clinic (92 families) and National Cancer Institute (NCI, 40 families), including 251 CLLs, 74 MBLs, and 246 unaffected relatives. DNA was extracted from buccal cells (Mayo Clinic) and peripheral blood (NCI). Allele counts from gVCF files were extracted for specific genes and analyzed. Variants were annotated using the Variant Effect Predictor in Ensembl, genome build GRCh37 (http://useast.ensembl.org/index.html). Highly-sensitive flow cytometry of the blood was used to screen for MBL. CHIP was defined as in Genovese et al., 2014 as the presence of any of the following: A) disruptive mutations in ASXL1, TET2, or PPM1D; B) missense mutation p.617F in JAK2; C) disruptive or missense mutations in DNMT3Alocalized to exons 7 to 23D) any mutations reported at least 7 times in hematological malignancies in the Catalogue of Somatic Mutations in Cancer (COSMIC); and (E) No variant allelic fraction (VAF) threshold. CLL driver genes included disruptive or missense mutations in ATM, BIRC3, MYD88, NOTCH1, SF3B1, TP53, EGR2, POT1, NFKBIE, XPO1, or FBXW7with VAF greater than 0.1. Mutations from COSMIC and CLL driver genes were excluded if the minor allele frequency in 1000 Genomes was greater than 0.5%. Data was analyzed using logistic regression adjusting for age, sex, and site. Odds ratios (OR) and 95% confidence intervals (CI) were estimated. Median age for CLL, MBL, and unaffected was 63, 61.5, and 54, respectively. The unaffected relatives were significantly younger than both the CLL and MBL groups (p<0.0001), with no difference between CLL and MBL (p=0.46). A total of 70 (12%) individuals from 54 different families had CHIP. CHIP was characterized in the majority of samples as mutations in DNMT3A, ASXL1, and TET2with 43, 9 and 8 individuals, respectively. As expected, CHIP was associated with age (p<0.001) but not sex (p=0.87). CHIP was present in 42 (17%) CLLs, 9 (12%) MBLs, and 19 (8%) unaffected relatives. There was a significant association between the presence of CHIP and CLL risk compared to unaffected relatives (OR= 2.19, 95% CI: 1.20 - 4.13, p=0.013) but not with MBL risk (OR=1.34, 95% CI: 0.54 - 3.11, p=0.51). Using the CLL driver genes, we found mutations in 119 (47%) CLL, 20 (27%) MBL, and 70 (29%) unaffected relatives. Mutations in CLL driver genes were associated with CLL risk compared to unaffected relatives (OR = 2.51, 95% CI: 1.64 - 3.87, p<0.0001), but there was no evidence for increased MBL risk (OR = 1.00). These results held when we subset the analysis to the Mayo only data where the DNA was extracted from buccal cells. This study further supports the association between mutations in CHIP related genes and risk of hematologic malignancies, in particular CLL. This is the first report of the prevalence of CHIP in MBLs. Although MBL and CHIP represent clonal population of blood cells, our data suggest the clones are different. Mutations in CLL driver genes are also associated with CLL risk but show little to no evidence with increased MBL risk. Interestingly, our unaffected relatives of CLL cases also had a high frequency of CLL driver mutations. Future studies are needed to understand these acquired mutations (both in CHIP- and CLL driver- related genes) in the unaffected to MBL to CLL continuum. Disclosures Parikh: Abbvie: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding; Gilead: Honoraria; Janssen: Research Funding. Ding:Merck: Research Funding. Cerhan:Jannsen: Other: Scientific Advisory Board; Celgene: Research Funding; Nanostring: Research Funding. Shanafelt:Jansen: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding; GlaxoSmithKline: Research Funding. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Abstract Backgrounds B cell receptor (BCR) signals and microenvironment protection have been recognized to have key influences on CLL survival and activation. Akt activation is down-stream of the BCR signaling cascade and is critical in the mediation of the bi-directional interactions between CLL B-cells and bone marrow stroma. MK2206 is an allosteric Akt inhibitor previously tested for safety in solid tumor patients. Our preclinical data has demonstrated the synergy of MK2206 with bendamustine (B) to induce CLL apoptosis in vitro. MK2206 also abolished the signal activation and cytokine production induced by BCR ligation in CLL leukemic cells (Ding, ASH 2012). Therefore, we conducted a phase 1 trial testing the safety and efficacy of MK2206 in combination with bendamustine and rituximab (BR) for patients with relapsed/refractory CLL. Methods A phase 1 dose-escalation study was designed to define the maximum tolerated dose (MTD) as well as the safety and efficacy of 3 dose levels (90 mg or 135 mg or 200 mg weekly) of the oral Akt inhibitor MK2206 in combination with bendamustine (70 mg/m2 daily for 2 days in each cycle) and rituximab (cycle 1: 375 mg/m2, cycle 2-6: 500 mg/m2) therapy in relapse/refractory CLL patients. To test the targeting efficacy of MK2206 on Akt and downstream signals, we designed a one-week “run-in” of single agent MK2206 before the initiation of BR during cycle 1. MK2206 was then given along with BR on day 1 of cycles 2-6. Tumor response was evaluated 2 months after the 6th/last cycle of therapy based on IWCLL 2008 criteria. Correlative laboratory studies included assessment of Akt signaling and the plasma cytokine levels (multiplex beads assay (Invitrogen)) at baseline and after one week of first dose of single agent MK2206. Results Nine patients (median age 68) were enrolled in the phase I portion of this trial. All 9 patients had unmutated immunoglobulin heavy chain variable region genes (IGHV) and 4 had del(11q). Six had received prior chemoimmunotherapy (CIT: FCR or PCR or PCO) and 5 had high risk disease defined as early progression within 26 months of the last CIT. Three had received alemtuzumab containing regimen. We have observed potent lymphocyte mobilization in all evaluated patients (n=7) after one-dose of MK2206 during week 1 (mean increase ALC: 35%) as well as reductions of plasma CCL3, CCL4 and CCL2 in the majority of the patients. A single dose-limiting toxicity (DLT) (febrile neutropenia and hemolytic anemia) was observed at dose level 1 (90 mg weekly) among 6 patients treated at this dose. Two patients with dose-limiting toxicities at dose level 2 (135 mg weekly) were observed where one patient had a rash and one patient had dehydration and febrile neutropenia among three patients treated at this dose. Accordingly, the 90 mg once weekly dose was determined to be the MTD for phase 2 trial testing. The most frequent grade 3 or 4 adverse events observed to date were neutropenia (44%) including febrile neutropenia (22%), rash (22%), diarrhea (22%), nausea and vomiting (11%), dehydration (11%), hemolytic anemia (11%) and thrombocytopenia (11%). The frequency of cytopenias was similar to the published German experience with BR (50% grade 3 or 4 hematological toxicities compared to 44% in N1087) in relapsed CLL patients (Fischer, JCO 2011). The median number of cycles received was 6 (range 1-6). Using an intent to treat analysis, two patients achieved complete response (CR, n= 2, 22%), one patient achieved nodular partial response (nPR, n=1, 11%) and five patients achieved a partial response (PR, n=5, 56%). The overall response rate (ORR) is 89%. Conclusion The Akt inhibitor MK2206 in combination with BR is well tolerated in patients with relapsed/refractory CLL. Single dose of MK2206 inhibits Akt phosphorylation, mobilizes leukemic cells and decreases plasma cytokines involved in BCR signals. The preliminary efficacy of this combination demonstrated promising results with a 89% ORR and 22% CR albeit in the small phase 1 cohort. These results compare favorably to BR alone (9% CR and 59% ORR) in relapsed/refractory CLL patients. Phase 2 testing of this combination is now underway. Acknowledgment This work was supported by the funding from NCI grant K23CA160345 (WD), NCI grant CA95241 (NEK) and Alliance for Clinical Trials in Oncology (CA025224 and CA33601). Disclosures: Off Label Use: MK2206, used in a phase 1 trial of relapsed CLL disease. Shanafelt:Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding. Zent:Novartis: Research Funding; GlaxSmithKline: Research Funding; Biothera: Research Funding; Genzyme: Research Funding; Genentech : Research Funding. Reeder:Celgene: Research Funding; Novartis: Research Funding; Millenium: Research Funding.

Abstract Abstract 4152 BACKGROUND: Patients with biologically aggressive chronic lymphocytic leukemia (CLL) characterized by being refractory to purine nucleoside analogues and/or possessing the cytogenetic abnormality del (17p13) have poor clinical outcomes with a median survival of <3 years. Several groups have reported that nonmyeloablative allogeneic stem cell transplant (alloSCT) provides long-term disease control in a subset of high-risk CLL patients. The Seattle group also recently reported that alemtuzumab use prior to alloSCT may increase the risk of early relapse/progression (Sorror, ASH 2010). This is a potentially critical observation since alemtuzumab is one of the few active agents in patients with del(17p13) and is frequently used for pre-transplant debulking in patients with this genetic defect. METHODS: We performed retrospective analysis of 38 CLL patients who underwent nonmyeloablative alloSCT for CLL at the Mayo Clinic between 2003 and 2011. The relationship between transplant outcome and debulking regimen used immediately prior to alloSCT, disease status at the time of alloSCT, post transplant engraftment, GVHD development was explored. The degree of marrow involvement prior to alloSCT was assessed using the leukemia index calculated by multiplying the % involvement by CLL cells and the % cellularity. RESULTS: Median age at the time of alloSCT was 55.5 (range, 29–69) and 13 patients (34%) were female. The median duration from CLL diagnosis to alloSCT was 4.7 years. Thirty-two patients (84%) had received more than 3 prior therapies prior to alloSCT. Thirty-seven patients (97%) had received purine analog-based therapies, 18 (47%) patients had received alkylating agents and 25 (66%) had received alemtuzumab. 33 (87%) patients were purine analog refractory. On cytogenetic evaluation by FISH, 17 (45%) patients had del(17p13), 10 (26%) had del(11q23) and 1 (3%) had del(6q23). Nine patients were in complete remission (CR) by IWCLL 2008 criteria at the time of alloSCT and 7 patients were in complete remission with incomplete marrow recovery (CRi). Of the remaining 22 patients, 20 had either a partial remission or stable disease at the time of alloSCT and 2 had progressive disease. Two (5%) patients had bulky lymphadenopathy (≥ 5cm by CT imaging), 11 (29%) patients had splenomegaly, and 8 (21%) patients had more than 30% CLL involvement by the leukemia index. Fludarabine (F) and 200cGy total body irradiation (TBI) was used as the conditioning regimen for 35 patients (92%). 22 patients received unrelated donor grafts (15 matched, 7 mismatched), and 16 received related donor grafts (all matched). Patients received mycophenolate (MMF) and either tacrolimus (unrelated grafts) or cyclosporin (related grafts) for graft versus host disease (GVHD) prophylaxis. 31 out of 37 evaluable patients (84%) achieved initial 100% donor chimerism (22 within 100 days) and 24 (63%) developed ≥grade 2 GVHD. After median follow-up of 1 year (range = 0–5.4 years), 26 of 38 patients (68%) are alive, and the median overall survival is 47 months. To date, 13 (34%) have experienced CLL progression/relapse requiring accelerated taper of GVHD prophylaxis (n = 2), donor lymphocyte infusion (n = 5), or chemo/immunotherapies (n= 6). For the 12 deceased patients, causes of death was CLL relapse/progression (n = 5), GVHD (n = 2) and infectious or other complications without CLL relapse/progression (n = 5). Pre-transplant use of alemtuzumab (A) within 3 months of transplant was the only pre-transplant characteristics associated with overall survival (OS). Median OS of patients receiving A within 3 months of alloSCT was 1 year compared to not reached for those who did not receive A within 3 months of alloSCT (p = 0.011; Figure 1a). A trend was also observed between alemtuzumab use within 12 months of alloSCT; however, it did not reach statistical significance (p=0.069; Figure 1b). No statistically significant associations between residual marrow involvement at time of alloSCT (p = 0.93), type of graft (p = 0.65), or presence of del (17p13) (p = 0.13), number of prior treatments (p = 0.45), patient age (p = 0.80) or disease status at time of alloSCT (p = 0.21) were observed. CONCLUSION: In this cohort of patients with high risk CLL, alemtuzumab use prior to transplant was the greatest predictor of OS after nonmyeloablative alloSCT. Non-alemtuzumab based debulking strategies need to be developed for patients with del (17p13). Disclosures: No relevant conflicts of interest to declare.

Abstract It is believed that malignant cells “condition” the microenvironment to facilitate tumor cell survival. We hypothesized that crosstalk between CLL B-cells and marrow stromal cells impacts both cell types bi-directionally and ultimately contributes to leukemic cell apoptotic resistance. To test this hypotheses, bone marrow stromal cells from core bone biopsies from CLL patients were isolated and cultured using methods we have previously described (Leuk Res 2007 31(7):899). Subsequently, we determined the impact of co-culture on CLL B-cell features including apoptosis and CD38 expression. In addition, we evaluated the release of angiogenic cytokines on co-culture and signal events in the stromal cells. Immunophenotyping demonstrated that cultured bone biopsy derived stromal cells were CD73+, CD105+, CD146+, CD14−, CD45−, CD34−, HLA-DR-, suggesting they were mesenchymal stem cells (MSC). Co-culture of these MSC with CLL B-cells protected CLL B-cells from both spontaneous apoptosis (SA) and drug-induced (fludarabine and chlorambucil) apoptosis (DA). For SA, the mean survival of CLL B-cells with or without co-culture of MSC for 5 days were 56.9 ± 10.0 and 7.7 ±3.7 (p<0.05), respectively. When CLL B cells were treated with fludarabine or chlorambucil, the fraction of CLL cells tightly adherent to MSC (TA-CLL) showed higher survival than a less adherent but viable fraction of CLL B-cells. The mean survival of TA-CLL cells treated with 10 μM of fludarabine for 48 hours in the presence of MSC were 67.5 ± 3.6 vs 29.8 ± 11.1 without MSC (P<0.05), respectively. When CLL cells with evidence for CD38 expression were co-cultured with MSC, both the percentage of CD38 positive cells and level of expression of CD38 per cell were up-regulated (mean fold change: CD38 percentage, 2.7, p<0.05; CD38 MFI, 1.9, p<0.05) after 2 weeks. In contrast, the CD38 percentage and expression were not changed in cells with minimal CD38 expression when these CLL B-cells were co-cultured with MSC. In addition, co-culture of MSC with CLL cells induced rapid ERK and AKT phosphorylation (within 30 min) in the MSC on immunoblot analysis. When CLL B cells and MSCs were cultured in transwells, the activation of ERK and AKT in MSC occurred at similar levels, indicating that activation of MSC was mediated by soluble factors. In addition, co-culture led to increased secretion of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) as well as a decrease of thrombospondin-1 (TSP-1) in the culture medium. These findings confirm that co-culture of CLL B-cells and MSC culminates in “angiogenic switch.” Taken together, these results strongly suggest interactions between MSC and CLL B cells are a bi-directional process. In leukemic cells, the interaction not only protects against spontaneous and drug induced apoptosis but also leads to an increase in CD38 expression consistent with an activated status. In MSC, the interaction leads to activation of ERK and AKT. Co-culture also facilitates angiogenic switching. These results underscore the dynamic and complex nature of the interactions between bone marrow stromal cells and CLL B-cells. Further studies are needed to dissect how crosstalk between CLL B-cells and MSC relates to disease progression, and determines whether these interactions can be targeted with therapeutic intent.

Abstract Abstract 3872 Background: Mesenchymal stromal cells are one of the components in the CLL microenvironment known to support leukemic cells survival and growth. Crosstalk between CLL B-cells and marrow-derived mesenchymal stromal cells (MSC) can modulate the activation of both cell types. Indeed we have shown that soluble factors secreted from CLL B cells can activate MSC with increased production of angiogenic cytokines (Blood. 2010. 116:2984). We hypothesize that MSC derived from CLL patients are modified by their interaction with leukemic cells in their gene expression profiles (GEP) and cytokine production (CP) capabilities. Given this, intrinsic differences should be present amongst MSC derived from CLL patients versus normal subjects. Method: MSC collected from CLL patients with Rai stage 1–4 before any therapy or who have not had therapies for at least 2 years as well as normal MSC from healthy controls were cultured and expanded one time in vitro. Subsequently, MSC after one in vitro passage were sorted with CD45 negative selection to eliminate any contaminating non-stromal cells. Approximately 100,000 to 300,000 cells were then stored at −70C and used for RNA purification. Nine CLL MSC and five normal MSC cell samples had RNA purified from each sample and then the RNA preparations were used for comparative analysis of GEP using affymetrix U133A plus B chip. Data were transformed using two different methods: MAS5 or Plier and only probes that were present in at least 50% of cases were included in the analysis. We have recently observed that MSC derived from bone marrow of CLL patients promotes Th17 cell differentiation (Blood. 2010. 116: Abstract 2414). Since IL-17 is the signature cytokine produced by TH17 cells, we decided to test if IL-17 could alter the cytokine production from CLL and normal MSC. Cytokine production from both CLL MSC and normal MSC as secreted into culture medium (CM) spontaneously or with recombinant IL-17 during a 72h-culture were tested using a commercial cytokine array (R&D). Result: Both MAS5 and Plier method of transformation yielded similar results for GEPs of the MSC. The preliminary data from these analyses showed that multiple transcription factors including PITX2, HOXB7, SIM1, ZFY, EMX2, HOXB3, HOXB4, and IRF6 were significantly up-regulated (more than 2 fold) in CLL MSC compared to normal MSC. In contrast, the genes up-regulated in normal MSC compared to CLL MSC include genes involved in the immune response (IGH, IGK, IGL, BST2, PPBP, RGS1, etc.), antigen processing and presentation (HLA genes, IFI30, etc.) and inflammatory response (CD163, LYZ, TNFAIP6, etc.). We are currently in the process of performing further analysis using additional MSC samples to validate these findings with further GEP assays and also by validating the dysregulated genes detected by our GEP studies using real-time PCR analysis. We performed the analysis of multiple cytokines produced by CLL (n=16) and normal MSC (n=12) with and without stimulation with recombinant IL-17 (8 ng/ml) over a 72 hour culture period. Without stimulation, there is approximately two-fold increase of MCP-1 production in CLL MSC compared to normal MSC (CLL vs. normal, 164.2 ± 26.1 vs. 82.7 ± 15.4 pg/ml, p = 0.02). After IL-17 stimulation, an approximately four-fold increase of MCP-1 production (CLL vs. normal, 1200.1 ± 258.6 vs. 334.1 ± 68.2 pg/ml, p = 0.005), three-fold increase of IL-8 production (CLL vs. normal, 2073.1 ± 504.1 vs. 708.7 ± 218.9 pg/ml, p = 0.02), two-fold increase of IL-6 production (CLL vs. normal, 1400.6 ± 214.0 vs. 705.6 ± 98.2 pg/ml, p = 0.008) were detected in CLL MSC compared to normal MSC. Conclusion: We have detected the presence of intrinsic mRNA expression differences for both transcription factors and immune response genes between CLL and normal MSC. The expression differences of these genes could be facilitating the differential cytokine responses we observed in CLL MSC compared to normal MSC. Given these findings it is possible that the dynamic interactions between the CLL leukemic cell and MSC influence the transcription profiles and cytokine production from MSC. This is of biologic and clinical interest in that the GEP dysregulation in CLL MSC may be a primary mechanism for the differential cytokine production seen in CLL MSC. This outcome in turn has the potential to modify the immune and non-immune environment in favor of tumor proliferation and progression. Disclosures: Kay: Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity's Board of Directors or advisory committees.

Abstract Background:Recurrent mutations in ATM, JAK1/3 and STAT5B genes have been identified in T-cell prolymphocytic leukemia (T-PLL), a mature T cell leukemia with poor survival. However, much remains to be understood regarding the dysregulated pathways involved in the pathogenesis of this aggressive disease. Here, we integrated the analyses of whole exome sequencing (WES) and transcriptomes of a cohort of 12 T-PLL samples to gain further insights into molecular mechanisms of this malignancy. Methods: Leukemic cells were isolated from peripheral blood or bone marrow samples of T-PLL patients. Tumor DNA and RNA, and germline DNA were extracted from purified leukemic T cells and bone marrow derived stromal cells, respectively. WES (n=12) and transcriptome analysis (n=10) were performed and data analyzed with published standard methods (Robinson et al. 2010 and DePristo et al. 2011) Results: 7 paired tumor and germline DNA and 5 tumor-only DNA samples were used for WES analysis. Mutations in ATM and TET2 were detected in 4 (33%) and 1 (8%) patients, respectively. JAK3 (p.M511I, p.Q501H and p.Q503H) or STAT5B (2 p.T628S and 1 p.N642H) mutations each occurred in 3 (25%) cases and were mutually exclusive, consistent with prior reports. Importantly, we found several novel mutations in genes with regulatory roles in the NOTCH signaling pathway. Specifically, 2 somatic mutations p.A3V and p.A3S in NOTCH2 and 2 other mutations (p.S244L and p.A1414V) in NOTCH4 were identified in 4 (33%) patients. One mutation was in the EGF- like and another one in the NODP domain critical for NOTCH-mediated cellular differentiation. Two (16.7%) novel missense mutations (p.G174D and p.V836I) were found in MAML2. Three missense mutations (2 p.S583N and 1 p.R190H) occurred in MAML1. Both MAML2 and MAML1 are essential cofactors of the NOTCH signaling pathway. In addition, a somatic mutation in the NOTCH signal regulator DTX2 (p.G268D) was detected. Altogether 7 (58.3%) patients harbored mutations in genes with a regulatory role in the NOTCH pathway. Among the epigenetic modifiers, we found deletions and point mutations in KDM6B and KDM6A in 4 (30%) patients including 2 codon deletions in KDM6B (p.X751Val) and 1 missense mutation each in KDM6B (p.R1016Q) and KDM6A (p.V510G). KDM6A and KDM6B are histone H3 lysine 27 demethylases known to be involved in regulation of DNA repair and play an important role in embryonic differentiation. Transcriptome analyses of 10 T-PLL cases versus normal CD3+ T cells from 5 healthy donors revealed 706 up-regulated and 655 down-regulated genes. Consistent with prior data, TCL1A and TCL1B were significantly overexpressed (23.7 and 13.1 fold, respectively). Using Ingenuity Pathway Analysis, we found PTEN Signaling, glutamate receptor, and axonal guidance signaling to be among the top 3 upregulated pathways. The top upstream regulators implicated for the up-regulated signals were FGF, Wnt/β-catenin and TNF. The top 3 down-regulated pathways were granulocyte adhesion and diapedesis, IL-17A in psoriasis and IL-10 signaling. The top regulators for the down-regulated pathways were LPS, TNF and IFNg. Wnt/β-catenin and Human embryonic stem cell pluripotency signaling were among the top 20 up-regulated pathways. Many genes involved in the regulation of Wnt signal pathway were included in the above pathways, including Wnt ligands (Wnt9A, Wnt4), transmembrane receptors (CDH1, LRP5, FZD6, FZD7, SMO), cytoplasmic factor (AXIN2), and nuclear factors (MYC, SOX12, SOX8, SOX18, CCND1). Summary: Our study identified recurrent mutations in the NOTCH signaling pathway as well as dysregulated embryonic developmental pathway, e.g. Wnt/NOTCH as newly implicated molecular mechanisms in T-PLL pathogenesis for further functional studies. Additionally, novel (KDM6A and KDM6B) and known mutations (ATM, TET2) in chromatin regulatory genes underscore the importance of epigenetic dysregulation in the tumorigenesis of T-PLL. Disclosures Parikh: Pharmacyclics: Honoraria, Research Funding. Mustjoki:Ariad: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Ding:Merck: Research Funding.