ALBERT

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: Abstract 600 Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a short median survival despite multimodal therapy. FTY720, an immunosuppressive drug approved for the treatment of multiple sclerosis, promotes MCL cell death via down-modulation of phospho-Akt and Cyclin D1, and subsequent cell cycle arrest (1). However, the mechanism of FTY720-mediated MCL cell death remains to be fully clarified. Here we show features of autophagy blockage by FTY720 treatment, including accumulation of autolysosomes, increased LC3-II and p62 levels. FTY720 is phosphorylated in vivo by sphingosine kinase 2 and converted to p-FTY720, which binds to sphingosine-1-phosphate (S1P) receptors. A non-phosphorylatable FTY720 derivative (OSU-2S) was recently developed at the Ohio State University (2): OSU-2S treatment induces MCL cell death and shows features of autophagy blockage that led us to conclude that FTY720 phosphorylation and its interaction with SP1 receptors are not required for FTY720-mediated cell death and blockage of autophagy in MCL cells. We also demonstrate that FTY720-induced cell death is mediated by lysosomal membrane permeabilization with subsequent translocation of lysosomal hydrolases to the cytosol. FTY720-mediated disruption of the autophagic-lysosomal pathway led to increased levels of CD74, a potential therapeutic target in MCL that is degraded in the lysosomal compartment. We have recently reported CD74 to be expressed on MCL cells and that milatuzumab (Immunomedics, Morris Plains, NJ), a humanized anti-CD74 monoclonal antibody, has significant anti-MCL activity in vitro and in vivo (3). This finding provided the rationale for examining combination therapy with FTY720 and milatuzumab. The in vitro survival of 4 MCL cell lines treated with FTY720, immobilized milatuzumab, and the combination was determined at 24 hours by Annexin-V/PI staining and flow cytometry. Incubation of 4 MCL cell lines with FTY720 and milatuzumab (1 μg/ml) resulted in a statistically significant decrease in cell viability compared to either agent alone for each of the four cell lines (P〈 0.01), despite using FTY720 at concentrations lower than the LC50 previously published [Jeko-1 FTY720: 10 μM (LC50: 12.5 μM), Z-138 and UPN-1: 6 μM (LC50: 7.5 μM); Mino 3.75 μM (LC50: 7.5μM)] (1). Notably, combination treatment resulted in synergistic killing in cell lines derived from patients with blastoid variant MCL (Jeko-1, Z-138, UPN-1), despite the fact that both FTY720 and milatuzumab as single agents showed only modest activity. Incubation of primary tumor cells from 6 MCL patients (3 blastoid variant and 3 classic MCL) with FTY720 (2.5 μM, LC50: 5 μM) and miltauzumab induced an average 78.5% cell death compared to 47% of FTY720 treated cells and 50% the milatuzumab-treated cells (P=0.0005 and P=0.0014, respectively). To examine the in vivo activity of FTY720 and milatuzumab, a preclinical model of human MCL using the SCID (CB17 scid/scid) mouse depleted of NK cells was used. In this model, i.v. injection of 40×106 JeKo cells results in disseminated MCL 3 weeks after engraftment. The primary end-point was survival, defined as the time to develop cachexia/wasting syndrome or hind limb paralysis. Mice (n=10/group) were treated starting at day 15 post engraftment. Twenty control mice received either placebo (saline) or trastuzumab (15 mg/kg) treatment. The third group was treated with FTY720 (5 mg/kg) every day for 2 weeks via i.p injection. The fourth group received milatuzumab (15 mg/kg) every three days, via i.p. injection. The fifth group received the combination of FTY720 and milatuzumab. The median survival for the combination-treated group was 36 days (95% CI:31,36), compared to 28 days for the saline-treated mice (95% CI:24,31), 27 days for the trastuzumab-treated mice (95% CI:23,29), 31 days for the FTY720-treated mice (95% CI:28,32), and 33.5 days for the milatuzumab-treated mice (95% CI:23,34). The combination treatment significantly prolonged survival of this group compared to control groups (P

Description: The nuclear export protein XPO1 is overexpressed in cancer, leading to the cytoplasmic mislocalization of multiple tumor suppressor proteins. Existing XPO1-targeting agents lack selectivity and have been associated with significant toxicity. Small molecule selective inhibitors of nuclear export (SINEs) were designed that specifically inhibit XPO1. Genetic experiments and X-ray structures demonstrate that SINE covalently bind to a cysteine residue in the cargo-binding groove of XPO1, thereby inhibiting nuclear export of cargo proteins. The clinical relevance of SINEs was explored in chronic lymphocytic leukemia (CLL), a disease associated with recurrent XPO1 mutations. Evidence is presented that SINEs can restore normal regulation to the majority of the dysregulated pathways in CLL both in vitro and in vivo and induce apoptosis of CLL cells with a favorable therapeutic index, with enhanced killing of genomically high-risk CLL cells that are typically unresponsive to traditional therapies. More importantly, SINE slows disease progression, and improves overall survival in the Eμ-TCL1-SCID mouse model of CLL with minimal weight loss or other toxicities. Together, these findings demonstrate that XPO1 is a valid target in CLL with minimal effects on normal cells and provide a basis for the development of SINEs in CLL and related hematologic malignancies.

Description: Abstract 3498 During cell cycle progression, D class cyclins activate cyclin dependent kinases (CDK) 4 and 6 to phosphorylate and inactivate Rb, allowing E2F-1 mediated transcription of additional cell cycle genes including cyclin E to drive S phase entry. This critical pathway is nearly universally dysregulated in cancer, providing tumor cells a strong growth advantage and escape from normal mitotic control. Substantial research is being directed toward targeting this pathway in many cancer types, with some preliminary successes being achieved with pharmacologic inhibitors of CDK4/6. However the development of alternative strategies to block this pathway could potentially provide broad therapeutic benefit. A prime example of a tumor with a disrupted cyclin D axis is Mantle Cell Lymphoma (MCL), in which the t(11;14) translocation places CCND1, the gene for cyclin D1, under the control of an immunoglobulin promoter. This results in sustained cyclin D1 expression in tumor cells and concomitant Rb inactivation, S phase entry and cell division. MCL is a relatively uncommon subset of Non-Hodgkin Lymphoma, but accounts for a disproportionate number of deaths. Treatments are limited and relapse is nearly universal; thus, new treatment strategies are essential for this disease. Silvestrol is a structurally unique, plant-derived cyclopenta[b]benzofuran with potent in vitro and in vivo anti-tumor activity in several model systems including B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). Silvestrol inhibits the initiation step of translation by preventing assembly of eIF4A and capped mRNA into the eIF4F complex, leading to selective loss of short half-life proteins such as Mcl-1 and cyclin D1. We therefore hypothesized that silvestrol, through the depletion of cyclin D1, would demonstrate efficacy in MCL. Silvestrol showed low nanomolar IC50 values in the JeKo-1 (13 nM), Mino (17 nM) and SP-53 (43 nM) MCL cell lines at 48 hr (MTS assay; cell death confirmed by propidium iodide flow cytometry). This potency was similar in primary MCL tumor cells. Longer exposure times substantially improved the cytotoxicity of silvestrol assessed at 48 hr (approximately 50% effect achieved with a 16 hr exposure vs. 80% effect with a 24 hr exposure), suggesting that the cellular impacts of this agent increase with exposure time. Cyclins D1 and D3 were dramatically reduced in MCL cell lines with just 10 nM silvestrol at 16 hr (cyclin D2 was undetectable in these cells), with subsequent loss of Rb phosphorylation as well as cyclin E mRNA and protein, culminating in G1 cell cycle arrest. Similar to what we previously showed in CLL and ALL cells, silvestrol treatment under these conditions also caused loss of Mcl-1 protein with concurrent mitochondrial depolarization, although the exact mechanism of silvestrol-mediated cytotoxicity in these cells is still under investigation. In an aggressive xenograft mouse model of MCL, silvestrol produced a highly significant improvement in survival [median survival of vehicle vs. silvestrol treated mice (1.5 mg/kg every 48 hr) = 27 vs. 38 days; P

Description: Acute myeloid leukemia (AML) is a heterogeneous myeloid neoplasm with complex pathophysiology. The diversities of karyotype, genetic mutations and epigenetic aberrations in AML result in difficult risk stratification and treatment selection. Despite the application of standard chemotherapies and immunotherapies, many AML patients relapse at least in part due to the failure to eradicate AML leukemic stem cells (LSC). New therapies to target these cells would be immensely valuable. CD33 is a widely expressed myeloid marker present on the majority of AML cells, and CD33-targeted immunotherapies have shown promising results. However, the majority of LSC lack CD33 expression and are not eliminated with such agents. CD123, the alpha chain of the interleukin-3 receptor (IL-3R) heterodimer, is expressed on the majority of LSC and in many AML tumor cells. Several efforts to target CD123 to eradicate LSC have emerged. To date, however, each of these agents exhibited shortcomings that limited their development. SL-401 (from Stemline Therapeutics, NY) is a recombinant fusion protein consisting of human IL-3 and truncated diphtheria toxin. IL-3 dictates the specificity for CD123expressing cells, and the catalytic unit of diphtheria toxin upon internalization inhibits the translational machinery to initiate cell death. SL-401 induced potent, dose-dependent (10, 100, 1000 ng/ml) cytotoxicity in AML patient cells ex vivo, as observed by annexin V/propidium iodide staining. (N=16, 48hr trend p

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: Key Points BI 836858, an Fc-engineered anti-CD33 antibody, mediates autologous and allogeneic NK cell–mediated ADCC. Decitabine increases ligands for activating NK receptors potentiating BI 836858 activity, providing a rationale for combination therapy.

Description: Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a short median survival despite multimodal therapy. FTY720, an immunosuppressive drug approved for the treatment of multiple sclerosis, promotes MCL cell death concurrent with down-modulation of phospho-Akt and cyclin D1 and subsequent cell-cycle arrest. However, the mechanism of FTY720-mediated MCL cell death remains to be fully clarified. In the present study, we show features of autophagy blockage by FTY720 treatment, including accumulation of autolysosomes and increased LC3-II and p62 levels. We also show that FTY720-induced cell death is mediated by lysosomal membrane permeabilization with subsequent translocation of lysosomal hydrolases to the cytosol. FTY720-mediated disruption of the autophagic-lysosomal pathway led to increased levels of CD74, a potential therapeutic target in MCL that is degraded in the lysosomal compartment. This finding provided rationale for examining combination therapy with FTY720 and milatuzumab, an anti-CD74 mAb. Treatment of MCL cell lines and primary tumor cells with FTY720 and milatuzumab resulted in statistically significant enhanced cell death, which was synergistic in blastic variant MCL cell lines. Significant in vivo therapeutic activity of combination treatment was also demonstrated in a preclinical, in vivo model of MCL. These findings support clinical evaluation of this combination in patients with MCL.