ALBERT

Description: Chronic Lymphocytic Leukemia (CLL) is characterized by accumulation of clonally expanded population of CD5+ CD19+ B lymphocytes in peripheral blood and secondary lymphoid organs, with a majority of circulating cells in a non-dividing resting stage. However CLL is no longer considered a static disease that results from simple accumulation of long-lived lymphocytes, but rather, is a dynamic process with a birth rate of about 0.1-2% of the entire CLL clone per day. The Eµ-Tcl1 mouse serves as an excellent model for the development of CLL as they develop a CLL like disease by 9-13 months of age, due to overexpression of the oncogene, T cell Leukemia 1 (Tcl1), in B cells through the Ig VH promoter and Eµ enhancer (Bichi et al. PNAS. 2002). In an adoptive transfer model, intravenous or intraperitoneal injection of primary CD5+ CD19+ CLL cells from the Eµ-Tcl1 CLL mouse into recipient syngeneic mice leads to development of a CLL like disease within 3-5 weeks of transfer. We have characterized the growth of CLL cells in these mice by periodic submandibular bleeding, ultrasonography of spleen and flow cytometry. We find that Eµ-Tcl1 CLL cells express more Prostate apoptosis response-4 (Par-4), a pro-apoptotic tumor suppressor protein, than normal B-1 or B-2 cells in mice. Par-4 is silenced by promoter methylation in more than 30% of all cancers. Par-4 has been shown to be secreted and to induce apoptosis selectively in various types of cancer cells but not in normal cells. Although we find that Eµ-Tcl1 CLL cells constitutively secrete Par-4, they are resistant to Par-4 mediated apoptosis. We show that CLL cells have constitutively active B-cell receptor signaling and that inhibition of BCR signaling with FDA approved drugs (i.e. Dasatinib, Ibrutinib, and Fostamatinib) causes a decrease in Par-4 protein and mRNA levels and increases apoptosis. Interestingly, systemic Par-4 appears to inhibit CLL growth in vivo, since adoptively transferred Eµ-Tcl1 CLL cells grew better in Par-4 null mice, despite excellent Par-4 expression and secretion by the transferred cells. Thus there were three times more CLL cells in the bone marrow and twice as many in the spleens of Par-4 null mice compared to their wild type counterparts. We conclude that even though Par-4 is pro-apoptotic in the CLL microenvironment, intracellular Par-4 is either rendered inactive or is potentially pro-survival in Eµ-Tcl1 CLL cells. (Supported in part by NIH grants) Disclosures No relevant conflicts of interest to declare.

Description: Chronic lymphocytic leukemia (CLL), characterized by accumulation of CD5+CD19+sIgM+ B lymphocytes in peripheral blood and lymphoid organs, is classified into indolent and aggressive forms. Patients with indolent CLL generally survive 5 to 10 years and do not require treatment until severe symptoms, while those with aggressive CLL show resistant to standard treatment and survive less than 24 months. While emerging B cell antigen receptor directed therapies are promising, resistance to such therapies pose problems warranting novel therapeutic approaches. MicroRNA (miR) profiling revealed lower expression of miR-29b in aggressive CLL associated with survival, drug resistance and poor prognosis via its up-regulation of anti-apoptotic proteins myeloid leukemia cell differentiation protein 1 (Mcl1) and oncogenic T-cell leukemia 1 (Tcl1). Thus, specific overexpression of miR-29b in B-CLL cells could be a potential therapy for aggressive CLL patients. Despite the promise, short circulation half-life, limited cellular uptake and off-target effects on non-desirable tissues pose a challenge for miR-based therapies. To promote efficiency and specificity of miR-29b delivery, we developed neutral immunonanoparticles with selectivity to CLL via targeting tumor antigen ROR1, which is expressed in over 95% of CLL but not normal B cells. We optimized a novel 2A2-immunoliposome (2A2-ILP) recognizing surface ROR1 on primary CLL cell to promote internalization and miR-29b uptake (n=6, p=0.042*). About 20-fold increased uptake of miR-29b was achieved with 2A2-ILP-miR-29b formulation compared to control. Further ROR1 targeted delivery of miR29b resulted in significant downregulation of DNMT1 and DNMT3a mRNA and protein (n=3, DNMT1: p= 0.0115*; DNMT3a: p=0.0231*, SP1; p=0.0031**) in primary CLL cells and a human CLL cell line OSU-CLL. Consistent with the downregulation of DNMTs, decreased global DNA methylation was observed in OSU-CLL cell line one week post- treatment with 2A2-ILP-miR-29b (n=3, p=0.0003***). To further study the in vivo ROR1-targeting efficiency of 2A2-ILP-miR-29b, we used our recently described Eµ-hROR1x Tcl1 CLL mouse model that develops CLL like disease with human ROR1 antigen in leukemic CD19+CD5+ B cells. Using hROR1+CD19+CD5+ leukemic cell engraftment model, we showed significant in-vivo efficacy of ROR1-ILP-miR-29b formulation associated with a) decreased number of circulating leukemic B220+CD5+ cells b) reduced splenomegaly (p=0.0461*, 2A2-29b: n=9; PBS: n=8) c) with extended survival (p=0.0075**, 2A2-29b: n=9; IgG-29b: n=7; 2A2-SC: n=7; PBS: n=8). In summary, 2A2-ILP effectively delivered functional miR-29b, resulting in downregulation of DNMT1 and DNMT3a, reduction of hypermethylation and anti-leukemic activity. Ongoing studies are aimed at understanding miR-29b mediated in-vivo methylome reprograming using our novel hROR1xTcl1 transgenic mouse model and ROR1-targeted miR-29b delivery formulation. Figure 1. Figure 1. Disclosures Byrd: Acerta Pharma BV: Research Funding.

Description: Acute myeloid leukemia (AML) is an incurable disease with 5 year survival rates of 10% in patients over 60 years. Poor tolerance to chemotherapy, chemo resistance and high rate of relapse warrants less toxic and more effective regimens in AML. OSU-2S is a novel non-immunosuppressive derivative of FTY720, a sphingosine analogue. The promising in-vitro and in-vivo activity of OSU-2S against a number of leukemias and lymphomas, and other malignancies such as hepatocellular carcinoma, impelled us to evaluate the activity of OSU-2S in AML. The potent cytotoxic activity of the OSU-2S (5µM, 24hrs) in AML cell lines HL-60, MV411 and MOLM13 (n=3; HL-60: p=0.008; MV411: p=0.04; MOLM13: p=0.0094) encouraged us to evaluate the effect of OSU-2S in primary leukemic cells from AML patients. OSU-2S (5µM, 24 and 48 hrs) demonstrated significant cytotoxic activity against AML cells, including high risk FLT3-ITD mutated AMLs, (n=13, p

Description: The discovery of predominantly inactive phosphatases in a variety of cancers and the potential for phosphatase targeted therapy as an alternative to kinase inhibitors especially in situations where the efficacy of the kinase inhibitors are compromised due to resistance mechanisms attributed to mutations and single nucleotide polymorphisms of the drug targets prompted us to evaluate potential activators of phosphatases in chronic lymphocytic leukemia (CLL) and other B cell malignancies. We have recently identified cytotoxic activity of OSU-2S, a novel non-immunosuppressive FTY720 derivative and PP2A activator against CLL. OSU-2S induced cytotoxicity was associated with PKC dependent phosphorylation of Serine 591 (S591) of tumor suppressor phosphatase SHP1 and its nuclear translocation consistent with a potential role for S591 phosphorylation. Here in, we demonstrate the molecular mechanisms and a rational approach for developing this novel agent for preclinical and clinical studies. In-vitro kinase assay demonstrated OSU-2S increased activity of purified PKC directly (p

Description: Key Points Ibrutinib is the first clinically viable irreversible ITK inhibitor. Ibrutinib inhibits the formation of Th2 but not Th1 immunity.

Description: Abstract 775 In chronic lymphocytic leukemia (CLL), mounting evidence points to an aberrant tumor associated Th2 bias that drives leukemic cell immune evasion, promotes formation of a supportive niche microenvironment, and functionally cripples innate and adaptive immunity. The end result is a high incidence of infections which is the primary cause of mortality in CLL. This same Th2 bias is induced by many other types of cancer. Th2 CD4 T-cells are singularly dependent upon IL-2-inducible T-cell kinase (ITK) for activation whereas Th1 CD4 and CD8 T-cells have compensatory resting lymphocyte kinase (RLK) which conducts T-cell receptor activation even in the absence of ITK. Thus, a clinically viable ITK inhibitor would be ideal for targeting immune suppression associated with CLL and potentially other types of cancer. Unfortunately, no such therapeutic is currently available. Ibrutinib, a confirmed inhibitor of the Bruton's tyrosine kinase (BTK) that irreversibly blocks downstream B-cell receptor activation, has demonstrated outstanding clinical activity in phase I/II clinical trials resulting in durable remissions in CLL. Our studies unveiled a previously uncharacterized Th1 cytokine switch in ibrutinib treated CLL patients which could not be attributed to B-lymphocytes. This ibrutinib-induced Th1 T-cell skewing was confirmed using the EμTCL1 mouse model of leukemia. Such alterations in cytokine patterns were reminiscent of mouse studies in which genetic ablation of ITK subverted Th2 immunity, thereby potentiating Th1-based adaptive immunity. The striking homology between BTK and ITK combined with intriguing in silico docking studies and promising in vitro kinase inhibition profiles with ibrutinib led to the hypothesis that this could be the first clinically viable irreversible ITK inhibitor. Cellular probe assays confirmed that the active site of ITK was covalently blocked by ibrutinib at pharmacologically relevant doses. Our comprehensive molecular analyses of T-cell signaling confirmed this in the Jurkat cell line. We further confirmed both molecular and functional outcomes in primary and in vitro polarized Th1 and Th2 CD4 T-cells. We found that mutation of the ITK-Cys442 covalent binding residue for ibrutinib alleviated molecular inhibition. We also demonstrated that Th1 and CD8 T-cell restricted expression of RLK provides a compensatory platform for T-cell activation offering a molecular explanation for the selective outgrowth of cytotoxic Th1 biased immunity. We further confirmed this effect using T-cells directly derived from CLL patients. To demonstrate that ibrutinib-induced ITK inhibition had direct clinical relevance in the setting of CLL we utilized a novel listeriosis/leukemia mouse model. In this model we clearly demonstrated complete recovery of functional immunity and all ibrutinib treated mice survived a potentially lethal Listeria monocytogenes infection. Our results expose novel molecular insights into the mechanism of action of ibrutinib in the context of Th2-biased immunosuppressive leukemia. We also postulate that ibrutinib's irreversible ITK inhibitory effects may prove effective in a number of other autoimmune, inflammatory, and viral diseases, including influenza A and HIV/AIDS. Disclosures: Jaglowski: Pharmacyclics: Research Funding. Chang:Pharmacyclics, Inc.: Employment. Maddocks:Pharmacyclics: Research Funding. Buggy:Pharmacyclics: Employment, Equity Ownership. Byrd:Pharmacyclics: Research Funding.

Description: Abstract 188 BACKGROUND: Introduction of the anti-CD20 antibody rituximab has led to remarkable progress in the development of targeted therapies for CLL and other B-cell malignancies. Despite prolonging patient survival, therapies targeting CD20 have not been curative. In recent years, alternative targets for therapeutic antibodies have emerged. One of the most promising targets has been CD37, which is highly expressed on malignant B-cells in chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma. The recent interest in this target has led to the generation of novel anti-CD37 therapeutics that could benefit from more extensive preclinical evaluation. However, preclinical development of these agents has been limited by the absence of appropriate leukemia animal models that provide targets expressing human CD37 (hCD37). Here we describe the development and characterization of a transgenic mouse where CLL-like leukemic B-cells express hCD37 and aggressively transplant into syngenic hosts. We demonstrate the utility of this unique mouse model by evaluating the in vivo efficacy of IMGN529, a novel antibody-drug conjugate targeting hCD37 that consists of the CD37-targeting K7153A antibody linked to the maytansinoid DM1 via the thioether SMCC linker. METHODS: The hCD37 transgenic mouse (hCD37-Tg) founder lines were generated by conventional methodology at the OSU Transgenic Facility. B-cell specific expression of hCD37 is driven by immunoglobulin heavy chain promoter and Ig-μ enhancer elements. Founder lines were evaluated by RT-PCR and flow cytometry to confirm RNA and protein expression, respectively. These lines were then crossed with the EμTCL1 mouse model of CLL to generate hCD37xTCL1 mice that develop CD5+CD19+hCD37+ leukemia. For in vivo studies, splenocytes from a leukemic hCD37xTCL1 donor were injected i.v. into healthy hCD37-Tg mice. Mice were randomly assigned to the following treatment groups (n=8–10 per group): IMGN529 conjugate, its K7153A antibody component, or negative controls (isotype antibody-DM1 conjugate or trastuzumab). Upon diagnosis of leukemia, a 10 mg/kg dose was administered i.p. and repeat doses were given 2 times per week for 3 weeks (70 mg/kg total). Peripheral blood disease was monitored by flow cytometry, using counting beads to obtain the absolute number of leukemic CD5+CD19+ B-cells. CD37 expression levels were determined by quantitative flow cytometry. In vitro cytotoxicity was evaluated after 24 hour incubation by flow cytometry with Annexin V and propidium iodide staining. RESULTS: IMGN529 and its K7153A antibody component demonstrated comparable in vitro activity against freshly isolated human CLL cells even in the absence of cross-linking agents (mean IMGN529 cytotoxicity=50.04% vs. 48.85% for K7153A; p=0.175; n=9). Both compounds also demonstrated cytotoxicity against hCD37 Tg B-cells ex vivo in a cross-linking dependent manner, and while expression of hCD37 in hCD37-Tg animals was B-cell specific, the expression levels were substantially lower than those observed in human CLL cells. In vivo studies with transferred hCD37xTCL1 splenocytes demonstrated rapid and complete depletion of CD5+CD19+ leukemic B-cells in response to IMGN529 conjugate, but not K7153A antibody treatment. After 1 week of IMGN529 treatment, peripheral blood leukemia was nearly undetectable and previously detected massive splenomegaly was no longer palpable. In contrast, leukemic counts and spleen sizes continued to increase in control cohorts. CONCLUSIONS: In summary, our group has generated a mouse model that develops a transplantable CD5+CD19+ leukemia expressing hCD37. We demonstrate the utility of this model for both in vitro and in vivo testing of therapeutics targeting hCD37. In addition, preclinical mouse studies expose the robust anti-leukemic effects of IMGN529 in this in vivo model of aggressive B-cell malignancy, despite the relatively low expression of hCD37 on the leukemic B-cells. Our engraftment model shows that IMGN529 is capable of eliminating widespread and highly proliferative mouse leukemia by a mechanism that is both CD37 antigen and conjugate dependent. Therefore, we propose that this novel therapeutic may also exhibit substantial efficacy in a wide range of human B-cell malignancies, even those with relatively low CD37 expression. [This work was supported by NIH (NM, JCB), LLS (NM, JCB) and Pelotonia (KAB)]. Disclosures: Deckert: ImmunoGen Inc.: Employment.

Description: Abstract 2499 Despite development of numerous chemotherapeutic agents against Chronic Lymphocytic Leukemia (CLL), drug resistance remains impediment in the successful treatment of CLL. Currently several kinases including SYK, BTK, Pim and PI3K are being targeted with inhibitors for therapy in CLL. However, no phosphatase activation directed therapeutics has been described for CLL. We have recently reported FTY720, a potent immunosuppressive agent derived from fungal sphingosine analog ISP-1 to exhibit potent in-vitro and in-vivo preclinical activity against CLL and Mantle cell Lymphoma through protein phosphatase 2A (PP2A) activation dependent mechanisms. However, the immunosuppressive nature of this drug hinders its further development for clinical therapy in cancer. To overcome this limitation we have developed several derivatives of FTY720 by structure-activity relationship analysis. Here we demonstrate OSU-2S as an agent that modulates SHP1 and PP2A protein phosphatases in CLL with potential therapeutic options. OSU-2S is a novel FTY720 derivative that does not interact with sphingosine 1 phosphate receptor 1, thus lacking immunosuppressive property without compromising the cytotoxic potential. Here we have shown preclinical activity of OSU-2S in cell lines representing CLL (MEC-1), ALL (697), lymphoblastic lymphoma cell (Raji, Ramos), Mantle cell Lymphoma (Mino, Jeko), T-cells (Jurkat) and primary CLL and T cell leukemia cells. In primary CLL B-cells, OSU-2S induces activation of major caspases including caspase 3, 8, and 9 resulting in Poly (ADP-ribose) polymerase cleavage. It also induces reactive oxygen species in CLL primary cells with no modulation of Bcl-2 or Mcl-1 levels. Interestingly, OSU-2S induces phosphorylation of Ser 591 of the SHP1 phosphatase in time dependent manner. Consistent with a previously described role for pSer-591 to serve as the nuclear localization signal for SHP1, OSU-2S induced accumulation of pSer-591 SHP1 in the nuclear fraction. Interestingly, OSU-2S induced phosphorylation of Ser-591 SHP1 appears to be mediated through PKC dependent mechanism as activation of PKC with PMA induced phosphorylation of Ser-591 SHP1 and PKC inhibitor Bisindolylmaleimide inhibited OSU-2S induced Serine 591 phosphorylation of SHP1. Moreover, concentrations of Bisindolylmaleimide that prevented the SHP1 Ser-591 phosphorylation also partially rescued OSU-2S induced apoptosis of primary CLL B cells. Co-immunoprecipitation experiments revealed association of PP2A with pSer-591 SHP1 in CLL cells treated with OSU-2S indicating a potential crosstalk between these two phosphatases. Consistent with this hypothesis OSU-2S treatment resulted in activation of PP2A in primary CLL B cells. Further, administration of OSU-2S into Eμ-Tcl-1 transgenic mice with high circulating peripheral blood leukemic cells resulted in significant reduction in circulating CD19+CD5+ leukemic B cells indicating therapeutic efficacy. Ongoing studies on the biochemical basis of cross-talk between the Serine threonine (PP2A) and protein tyrosine (SHP1) phosphatases in response to OSU-2S treatment in CLL will be presented. This work was supported by LLS-Specialized Center of Research in Leukemia and NCI-Leukemia SPORE Grants Disclosures: No relevant conflicts of interest to declare.

Description: Chronic lymphocytic leukemia (CLL) occurs in 2 major forms: aggressive and indolent. Low miR-29b expression in aggressive CLL is associated with poor prognosis. Indiscriminate miR-29b overexpression in the B-lineage of mice causes aberrance, thus warranting the need for selective introduction of miR-29b into B-CLL cells for therapeutic benefit. The oncofetal antigen receptor tyrosine kinase orphan receptor 1 (ROR1) is expressed on malignant B-CLL cells, but not normal B cells, encouraging us with ROR1-targeted delivery for therapeutic miRs. Here, we describe targeted delivery of miR-29b to ROR1+ CLL cells leading to downregulation of DNMT1 and DNMT3A, modulation of global DNA methylation, decreased SP1, and increased p21 expression in cell lines and primary CLL cells in vitro. Furthermore, using an Eμ-TCL1 mouse model expressing human ROR1, we report the therapeutic benefit of enhanced survival via cellular reprograming by downregulation of DNMT1 and DNMT3A in vivo. Gene expression profiling of engrafted murine leukemia identified reprogramming of cell cycle regulators with decreased SP1 and increased p21 expression after targeted miR-29b treatment. This finding was confirmed by protein modulation, leading to cell cycle arrest and survival benefit in vivo. Importantly, SP1 knockdown results in p21-dependent compensation of the miR-29b effect on cell cycle arrest. These studies form a basis for leukemic cell–targeted delivery of miR-29b as a promising therapeutic approach for CLL and other ROR1+ B-cell malignancies.