ALBERT

Description: Patients with Chronic Lymphocytic Leukemia (CLL) have a variety of chromosomal abnormalities and mutations. At diagnosis, about 10% of CLL patients have deletions of chromosome 17 (Del17p) leading to the loss of one allele of tumor suppressor protein TP53, which increases to over 30% in relapsed/refractory disease. Additionally, 83% of patients with a Del17p acquire a mutation on their second TP53 allele at one of several sites within the DNA binding domain. While the consequence of some of these "hotspot" mutations (R175H, R179H, G245D, G248Q/W, Y220, R213X, R273H and R282H) has been described in solid tumors and AML, very little is known of their role in CLL. Clinically, patients with Del17p/Mutp53 have worse overall survival, increased disease progression and are more likely to relapse on the current targeted therapies such as ibrutinib. Although relapse to these treatments is largely due to acquired mutations in Bruton's Tyrosine Kinase (BTK) or its downstream target PLCg2, we hypothesize that the biology of mutant 53 bearing CLL is a key driver of resistance and progression. Specifically, we aim to determine the molecular signature and downstream effectors that allow mutant p53 to drive the adverse biology associated with this subtype. Conversely, we hypothesize that targeting the mutant p53 pathway will lead to better outcomes and overall survival for patients bearing this adverse prognosis marker. We performed high-throughput Sequencing of DNA from 270 CLL patients with high coverage in the exonic regions of TP53 prior to ibrutinib therapy as well as during progression. At baseline, 40% of patients had mutations found in the DNA binding region with the most frequent occurring in R248Q, R175H and R273H. We then characterized each p53 mutant (n=106) functionally in terms of their ability to ability to activate p21, PUMA, and Bax which serve as cell cycle checkpoint and apoptotic effectors of wild type p53 in response to DNA damage. Most mutants were incompetent in upregulating p21, PUMA or Bax at the transcript level. A few mutants upregulated p21 protein in a p53 independent fashion. We then evaluated the consequence of mutant p53 in CLL. We performed chromatin immunoprecipitation (ChIP-Seq), open chromatin signatures (ATAC-Seq) and expression analysis (RNA-Seq) on CLL samples with R248Q or R175H as well as in wild-type (WT) p53 samples. Integration of ChIP, ATAC and RNA Seq profiles indicated that mutant p53 activated a unique transcriptomic profile not shared by wt p53 bearing CLL. Several genes that facilitated survival or progression were downstream targets of mutant p53. Of these, we identified PRKCB (PKC-beta), BCL2L1 (Bcl-xL), EZH2, MLL and MALAT as a potential key downstream effectors of mutant p53. To determine whether mutations at R248Q and R175H in p53 were causal in the observed increases in PKC-beta, Bcl-xL, EZH2, MLL, and MALAT we used CRISPR/Cas9 editing to introduce mutations at R175H and R248Q in the p53 wildtype CLL cell lines HG-3 and PGA-1. These were accomplished by electroporating sgRNA-Cas9 ribonucleoprotein complexes (RNPs). Western blotting of mutants revealed an increase in the mRNA and protein expression of PKC-beta, and BCL-xL in mutant p53 compared to WT. Levels of EZH2 and MLL were not increased in these cells indicating that PKC-beta and Bcl-xL may be direct transcriptional targets upregulated by mutations at R248Q and R175H in p53. Ongoing efforts will characterize the transcriptional profile of all p53 mutants in our cohort to determine whether they all have a unifying transcriptomic profile that confers a gain of function phenotype to this subtype of CLL. Disclosures Byrd: Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Novartis: Other: Travel Expenses, Speakers Bureau; Acerta: Research Funding; BeiGene: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Genentech: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau. Woyach:Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Research Funding; Karyopharm: Research Funding; Loxo: Research Funding; Morphosys: Research Funding; Verastem: Research Funding.

Description: Restoring nuclear localization of tumor suppressors by blocking exportin 1 (XPO1) holds promise as a new therapeutic paradigm in many cancers, including chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). Oral selective inhibitor of nuclear export (SINE) compounds that covalently modify XPO1 were recently discovered and are exciting new compounds to implement this strategy. Selinexor, the clinical lead SINE, has made progress in Phase I/II clinical trials and is generally well tolerated, but limited to twice weekly dosing, with supportive care. The discovery of novel SINE compounds with improved tolerability is therefore of considerable clinical interest and represents a significant contribution beyond the targeted therapies currently available for hematologic malignancies and a variety of other cancers where upregulation of XPO1 is observed. Presented herein is the discovery of KPT-8602, the next generation SINE compound that shows lower brain penetration, improved tolerability allowing continuous dosing, and improved efficacy beyond any current XPO1 inhibitor. Results: Our crystallography data revealed that KPT-8602 binds covalently to XPO1 through a Michael acceptor that is activated by an electron withdrawing pyrimidyl moiety, allowing a 2-fold increased reversible interaction with XPO1 compared to earlier SINE compounds. The crystal structure of the KPT-8602-XPO1 complex showed interactions between XPO1 and the activating pyrimidyl group. In vitro pull-downs using immobilized GST-nuclear export sequences and purified recombinant XPO1 demonstrated greater reversible binding of KPT-8602 compared to KPT-330 (selinexor). In vivo toxicology studies demonstrated that KPT-8602 possesses lower brain penetration compared to KPT-330 allowing for continuous dosing and improved tolerability. Our results also showed that KPT-8602 induced a comparable level of cytotoxicity as well as inhibition of cell proliferation compared to KPT-330 in primary CLL tumors and in a representative panel of DLBCL cell lines. Furthermore, KPT-8602 inhibited proliferation and induced apoptosis in AML cell lines and primary AML blasts while inducing nuclear accumulation of p53 and NPM1. We hypothesized that these improved pharmacological parameters would allow daily KPT-8602 to abrogate disease progression in CLL and AML animal models. The Eµ-TCL1-C57BL/6 transplant model of CLL was used to evaluate the therapeutic benefit of continuous dosing of KPT-8602. Eµ-TCL1-engrafted mice were treated with KPT-8602 given daily or 2x/week. The KPT-8602 daily cohort had significantly improved survival with a median overall survival of 70 vs 50 days (vs vehicle 33 days), compared to those treated only 2x/week with KPT-8602 (p=0.001). Mice treated with KPT-330 2x/week showed a similar survival to mice treated with KPT-8602 2x/week. Mice given daily KPT-8602 had significantly smaller spleens and reduced circulating leukemic cells compared to all the other groups (p

Description: Key PointsAEB071 demonstrates preclinical in vitro and in vivo activity against CLL independent of survival signaling and stromal cell protection. AEB071 can either inhibit or activate the WNT pathway emphasizing the importance of pharmacodynamic monitoring in its development.

Description: Epstein-Barr virus (EBV) is a human herpesvirus that infects over 90% of the world's population and is associated with a wide-range of diseases. EBV has evolved to manipulate host cellular networks in the absence of proper immune function and is linked to malignant lymphoproliferative disorders (LPD) such as Hodgkin's lymphoma and diffuse large B-cell lymphomas. The lack of standard or effective therapeutic approaches for individuals with these aggressive and clinically complicated diseases represents an important unmet need. We have recently demonstrated EBV-induced B cell transformation to be dependent upon the dysregulation of protein arginine methyltransferase 5 (PRMT5), an epigenetic writer that functions as a global transcriptional repressor. We hypothesize that epigenetic readers, writers and erasers are similarly dysregulated following EBV infection of B cells and that these enzymes represent novel therapeutic candidates. The bromodomain and extra-terminal (BET) protein bromodomain 4 (BRD4) is an epigenetic reader that binds acetylated lysine residues and promotes transcription of genes that drive cell growth and survival. Here, we evaluate the relevance of BRD4 to EBV-driven B cell transformation and identify it as a potential therapeutic target for EBV-LPD. We used the highly selective and potent BRD4 inhibitor (BRD4i), JQ1, as a model to examine the relevance of BRD4 in EBV-driven lymphoproliferative disease (LPD). JQ1 treatment of, EBV-transformed lymphoblastoid cell lines (LCLs) led to reduced proliferation, but not direct cytotoxicity. However, similar concentrations of JQ1 in a more physiologically-relevant co-culture setting of EBV-LCLs cultured with autologous peripheral blood lymphocytes (PBMC), resulted in a robust depletion of LCLs, loss of suppressive myeloid populations (TAM/M2 phenotype) and expansion of anti-tumor adaptive, memory CD3/CD8 immune effector cells. Using a single dose of JQ1 (500nM) in a co-culture of LCLs and autologous PBMCs, there was a marked expansion of activated, effector memory (CD3+, CD8+, CD45RO+, CD62L-) cytotoxic T-cell population as compared to vehicle-treated co-cultures. Furthermore, a dramatic reduction in both CD19+ LCLs and suppressive myeloid populations (CD33+, CD11b, HLADR+, CD206+, PD-L1+) was observed after 10 days. Despite the depletion of myeloid cell subsets by CD33 depletion, we still saw the outgrowth of cytotoxic T cell population in the presence of JQ1. In order to delve into the causes of this heightened immune response, we looked at the changes in the expression of highly immunogenic EBV specific proteins. BRD4 inhibition led to upregulation of the EBV oncoprotein, latent membrane protein 1(LMP1), as well as MHC class I antigen presentation machinery in LCLs. The enhanced expression of LMP1 in LCLs with BRD4i, led to modulation of downstream signaling networks driven by this oncogene including PI3K/pAKT (decreased), NF-ĸB (canonical down/non canonical up) and pSTAT3 (decreased). Because LMP1 oncogenic activity is vital for EBV-driven B cell immortalization and transformation, we next conducted in-vitro assays where primary, naïve B cells were infected with EBV in the presence and absence of JQ1 (50, 500nM) treatment. Selective BRD4i was introduced in separate transformation cultures in seven-day intervals following EBV infection and absolute cell counts were monitored. At all timepoints BRD4i prevented EBV-driven transformation of purified naïve B-lymphocytes. Our findings highlight the utility of BRD4i as an experimental therapeutic strategy for EBV-driven lymphomas as it functions to target pathways initiating and sustaining transformed B cell outgrowth and survival while supporting anti-tumor host memory, immune networks. Collectively, these experiments identify BRD4 as a key driver of cell cycle progression, oncogene activation, and a potential immune checkpoint modulator in transformed B lymphoblasts. Furthermore, BRD4i enhances host immunity by eliminating suppressive myeloid cell populations permitting the activation and expansion of memory CD3+/CD8+ cytotoxic T cells. This work demonstrates BRD4i is an attractive therapeutic strategy as it sensitizes malignant cells while enhancing the responsiveness of the host immune system, making it an ideal candidate to be used in future trials with T cell immune therapies. Disclosures Baiocchi: Essanex: Research Funding.

Description: Introduction Blocking Bruton tyrosine kinase (BTK) with ibrutinib has demonstrated efficacy in chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL) patients (pts). However, resistance to therapy is common. Preclinical data suggest selinexor (oral selective inhibitor of nuclear export) downregulates BTK (Hing 2015). We hypothesized that dual BTK blockade by combining ibrutinib and selinexor would be tolerable and efficacious in CLL/NHL pts. Herein, we report the phase I combination study with expansion cohorts. Methods Pts with histologically confirmed CLL/NHL were enrolled (n=33). Eligible pts were age ≥18 years (yrs), had ≥1 prior therapy and ECOG performance status of 0-1. CLL pts met iwCLL 2008 criteria for therapy. Pts received selinexor orally 1-2 times weekly (3 weeks of 4-week cycle) and daily oral ibrutinib (420mg) starting Cycle 1 Day 8. Treatment cycles were repeated until disease progression, intolerance, death or discontinuation of trial participation. Primary endpoint was determination of maximum tolerated dose (MTD). Dose-finding proceeded using a modified continual reassessment method targeting a probability of dose limiting toxicity (DLT)

Description: CD19 is a B cell lineage-specific transmembrane signaling protein that controls differentiation and proliferation. CD19 is an attractive therapeutic target due to its high level of expression in numerous B cell malignancies, as well as its lack of expression on non-B cells. Here we report the in vitro anti-tumor activity of a novel humanized monoclonal anti-CD19 Ab (CD19-IgG1, aka XENP5603) and its Fc engineered counterpart (XmAb™CD19, aka XENP5574). XENP5603 induced direct apoptosis in normal CD19+ B cells, but not NK cells, T cells, or monocytes, as determined by flow cytometric staining with annexin V and propidium iodide. XENP5603 also induced significant levels of apoptosis in a number of lymphoblastoid cell lines, including Ramos, Raji, 697, NALM6, and RS4;11 cells. Treatment of primary chronic lymphocytic leukemia (CLL) cells with XENP5603 induced significant cell death in all patients tested (mean, 36% apoptotic cells at 24 hours; range, 13–66%, p 〈 0.001). Similar apoptosis was noted in cells from a subset of patients (4 of 9) with CD19+ primary acute lymphoblastic leukemia (ALL). Apoptosis of CLL cells treated with XENP5603 was not associated with cleavage of caspase-3, caspase-8, caspase-9, or PARP, but was associated with upregulation of Bim, suggesting a caspase-independent mechanism of cell death. NK cells from normal donors exhibited high levels of ADCC in response to B cell lines coated with XENP5603. Furthermore, NK cells from CLL patients mediated significant ADCC against autologous CLL cells in the presence of XENP5603 (mean, 15% specific lysis at an E:T ratio of 25:1; range, 8–24%; p = 0.04 vs. the negative control Ab). ADCC activity was further increased in the presence of XENP5574, which has the same antigen-recognition sequences as XENP5603 but which contains two mutations in the Fc region that increase FcγRIIIa affinity (mean, 39% specific lysis at an E:T ratio of 25:1; range, 29–51%; p = 0.02 vs. the negative control Ab). ADCC mediated by either CD19 Ab was also significantly higher than that mediated by an equivalent concentration of rituximab (mean, 39% specific lysis with XENP5574 vs. 12% with rituximab; p 〈 0.001). ADCC in the presence of either Ab was further increased in the presence of the NK cell-activating cytokine IL-2, suggesting that these antibodies might be effectively combined with immune stimulatory adjuvants. Furthermore, NK cell ADCC against CLL cells in the presence of CD19 Abs was found to be dependent on perforin/granzyme release, as treatment with 3,4-dichloroisocoumarin (which inhibits granzyme enzymatic activity) or EGTA (which prevents release of cytotoxic vesicles) potently inhibited ADCC activity. Collectively, these studies provide evidence of the autologous innate immune-mediated cytotoxicity and direct apoptotic activity of XENP5603 and XENP5574. In addition, engineering to enhance FcγRIIIa binding enhances autologous ADCC, providing support for further pre-clinical development of XENP5574 in CD19+ malignancies, including CLL and ALL.

Description: Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a median survival of 3 years despite chemoimmunotherapy. Rituximab, a chimeric anti–CD20 monoclonal antibody (mAb), has shown only modest activity as single agent in MCL. The humanized mAb milatuzumab targets CD74, an integral membrane protein linked with promotion of B-cell growth and survival, and has shown preclinical activity against B-cell malignancies. Because rituximab and milatuzumab target distinct antigens and potentially signal through different pathways, we explored a preclinical combination strategy in MCL. Treatment of MCL cell lines and primary tumor cells with immobilized milatuzumab and rituximab resulted in rapid cell death, radical oxygen species generation, and loss of mitochondrial membrane potential. Cytoskeletal distrupting agents significantly reduced formation of CD20/CD74 aggregates, cell adhesion, and cell death, highlighting the importance of actin microfilaments in rituximab/milatuzumab–mediated cell death. Cell death was independent of caspase activation, Bcl-2 family proteins or modulation of autophagy. Maximal inhibition of p65 nuclear translocation was observed with combination treatment, indicating disruption of the NF-κB pathway. Significant in vivo therapeutic activity of combination rituximab and milatuzumab was demonstrated in a preclinical model of MCL. These data support clinical evaluation of combination milatuzumab and rituximab therapy in MCL.

Description: In patients with chronic lymphocytic leukemia (CLL), lenalidomide can promote humoral immune responses but also induces a distinct disease-specific toxicity of tumor flare and cytokine release. These CLL-specific events result from increased expression of costimulatory molecules on B cells. Here we demonstrate that lenalidomide activation of CLL cells depends on the phosphatidylinositol 3-kinase p110δ (PI3K-δ) pathway. Inhibition of PI3K-δ signaling by the PI3K-δ-inhibiting drug, CAL-101, or by siRNA knockdown of p110δ, abrogates CLL cell activation, costimulatory molecule expression, and vascular endothelial growth factor and basic fibroblast growth factor gene expression that is induced by lenalidomide. In addition, CAL-101 attenuates lenalidomide-mediated increases in immunoglobulin M production by normal B cells. Collectively, these data demonstrate the importance of PI3K-δ signaling for lenalidomide immune modulation. These findings may guide development of strategies for the treatment of CLL that combine lenalidomide with CAL-101, with other inhibitors of the PI3K-δ pathway, or with other agents that target downstream kinases of this signaling pathway.