ALBERT

Description: Key Points High miR-10 family expression levels in AML patients are associated with achieving complete remission to induction chemotherapy. Functional experiments did not show any impact of miR-10a-5p in AML blast growth or survival at baseline conditions or after chemotherapy.

Description: Abstract 2422 Today most AML patients (pts) still fail to achieve long-term survival. New therapeutic options are needed. We and others reported that high miR-181a expression associated with better outcomes in AML pts (Marcucci et al NEJM 2008; Schwind et al JCO 2010; Li et al Blood 2012), but the underlying mechanisms remain unclear. Aberrant RAS activation by mutations or overexpression is frequent in human malignancies, including AML. Previously KRAS was described as a direct miR-181a target in oral squamous cell carcinoma (Shin et al BBRC 2011). Here we confirmed KRAS as a direct miR-181a target & identified miR-181a-binding sites in the MAPK1 untranslated region (UTR) utilizing in silico tools & consequently identified MAPK1 as a new direct miR-181a target of the RAS signaling pathway. We observed respectively a 30±5% (±standard deviation; P=.003) & 35±2% (P=.0002) downregulation of luciferase activity after co-transfecting 293T cells with miR-181a mimic vs scramble control (sc) molecules and luciferase reporter constructs spanning the predicted miR-181a binding sites (seed sequences) in KRAS or MAPK1. Mutations in the seed sequences of KRAS or MAPK1 could rescue the miR-181a mimic induced downregulation. In the KG1a & OCI-AML3 AML cells with low endogenous miR-181a level but activated RAS-signaling, lentiviral miR-181a overexpression reduced KRAS protein by 2.8 & 2.1 fold and MAPK1 by 2.8 & 1.5 fold (normalized to β-actin & quantified by densitometry) respectively compared to sc expressing controls. Consistent with this result, lentiviral-based knock-down of miR-181a increased KRAS by 1.4 & 3.2 fold and MAPK1 protein by 2.3 & 1.8 fold in KG1a & OCI-AML3 cells respectively, compared to sc controls. Since higher miR-181a associated with improved outcomes & miR-181a targets the RAS-pathway in AML, increasing miR-181a may have therapeutic value for AML pts. We formulated transferrin (Tf) targeted anionic lipid based lipopolyplex nanoparticles (NP) to deliver synthetic miR-181a mimic molecules to AML cells which overexpress Tf receptors, while overcoming RNA degradation & facilitating the cellular uptake of the miR molecules. Mature miR-181a in KG1a, OCI-AML3 cells & pts blasts (n=3) were measured by qPCR after treatment with Tf conjugated NP (TfNP) encapsulating miR-181a mimic molecules (TfNP181a) or sc (TfNPsc) at 10 nM. After 24h mature miR-181a levels increased 211±31, 880±10 & 145±19 fold in KG1a, OCI-AML3 & pts blasts (n=3), while levels of miR-181b & unrelated miR-140 remained unchanged. We found that delivered miR-181a downregulated KRAS & MAPK1 proteins (KG1a: 2.5 & 2.1 fold; OCI-AML3: 2.5 & 2.4 fold; pts blasts: 9.0 & 5.8 fold respectively) compared to TfNPsc treatment. To analyze downstream effects of TfNP181a mediated inhibition of the RAS pathway we assessed the expression of total MEK & AKT and p-MEK & p-AKT. TfNP181a compared to TfNPsc treatment decreased p-MEK by 3.2, 2.1 & 5 fold and p-AKT by 2.9, 3.6 & 6.5 fold in KG1a, OCI-AML3 & pts blasts. Furthermore, a 3.2, 4.7 & 3.1 fold reduction of the transcription factor MYC, which is regulated by MAPK in KG1a, OCI-AML3 & pts blasts treated with TfNP181a vs TfNPsc was observed. TfNP181a treatment also resulted in antileukemic activity. TfNP181a compared to TfNPsc treatment led to reduced proliferation of KG1a (34±8% at day 4, P=.01) & OCI-AML3 (31±4% at day 4, P=.02) cells, and decreased colony formation (after 2 weeks) in both cell lines by 40±5% (P

Description: AML is one of the most common types of leukemia. Despite recent progress in understanding the mechanisms of leukemogenesis, molecular risk assessment and risk-adapted treatments, only 40% of younger (

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: microRNA-155 (miR-155) is a short non-coding RNA that is associated with aggressive cancers and known to promote leukemogenesis. Recently, we have reported that aberrant miR-155 upregulation independently identifies high-risk cytogenetically normal AML patients, suggesting that this miR may also serve as a novel therapeutic target in AML. We and others have shown that miR-155 is positively regulated by NF-kB, a transcription factor that is constitutively activated in leukemic blasts and contributes to their aberrant proliferation and survival. MLN4924 (Millennium Pharmaceuticals Inc) is a novel drug that blocks neddylation and subsequent degradation of the NFkB inhibitor, IkBa, thereby inhibiting translocation of NF-kB to the nucleus. MLN4924 has demonstrated promising activity in early clinical trials for AML. We postulated that downregulation of miR-155 via NF-kB inhibition is at least in part responsible for the antileukemic activity of MLN4924. Methods AML cell lines and primary blasts were treated with 100-1000nM MLN4924 for 3-72 hrs. Messenger RNA and protein levels were determined by quantitative RT-PCR and immunoblotting, respectively. NF-kB activity was measured by luciferase reporter assays. Binding of NF-kB to the miR-155 promoter was detected by electromobility shift assay and Chromatin Immunoprecipitation. Transfection of miR-155 was performed using the siPORT TM NeoFXTM method. Apoptosis was assessed by Annexin V staining. For in vivo studies, we used NOD/SCID/g mice engrafted with MV4-11 cells. Two weeks after transplantation, the engrafted mice received intraperitoneal treatments of 180 mg/kg of MLN4924 every other day for 21 days. Mice in the control group were treated similarly with the vehicle alone (20% 2-hydroxypropyl-betacyclodextrin). Results In AML cell lines and primary AML patient blasts 12hr treatment with MLN4924 resulted in a ∼50% decrease of miR-155 expression at 300nM in THP-1 and MV4-11 cells and at 500nM in AML blasts (p

Description: Abstract 81 Despite advances in our understanding of disease mechanisms the outcome of the majority of AML patients (pts) remains poor. Epigenetic gene silencing by DNA hypermethylation has been shown to contribute to AML. Recently we demonstrated promising results of treatment with the hypomethylating agent DAC in older AML pts. Pts with higher miR-29b expression were more likely to respond to DAC. MiR-29b targets DNA methyltransferases (DNMTs; i.e. DNMT1, DNMT3A & DNMT3B) and has hypomethylating and tumor suppressor activity in AML. Thus uncovering a method to increase intracellular miR-29b and sensitize AML blasts to DAC may be of therapeutic value. Free synthetic miRs are degraded in bio-fluid and have very limited cellular uptake. To overcome these limitations, we developed a novel non-viral delivery system for synthetic miRs. Polyethylenimine was used to capture synthetic miRs & form a polyplex core. An outside layer of lipid components, 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine, Linoleic acid & 1,2-Dimyristoyl-sn-glycerol, methoxypolyethylene Glycol (MW=2,000) was used to protect the miRs from degradation & clearance and facilitate their uptake. Using this approach we produced nanoparticles (NPs) encapsulating 0.4 μM synthetic miR-29b precursors (pM29b) or scramble controls (sc) [both Ambion]. Since AML blasts and cell lines overexpress the transferrin receptor, we conjugated the NPs with transferrin (Tf) to increase the uptake by targeted delivery. To evaluate the efficiency of NP uptake we encapsulated FAM-fluorescent labeled-pM29b and treated AML cell lines (i.e. Kasumi-1 & MV4-11). Tf-conjugated NP (TfNP) treated cells showed the highest uptake by confocal microscopy and flow-cytometry (mean fluorescence intensity (MFI): 21.0 & 22.9 for Kasumi-1 & MV4-11 respectively) compared to non-Tf-conjugated NPs (NP; MFI: 8.3 & 13.0). To evaluate if pM29b was processed into its mature form we measured levels of mature miR-29b in MV4-11 & Kasumi-1 by qPCR after treatment with TfNP encapsulating pM29b (TfNP29b) or NP encapsulating pM29b (NP29b). At 48h mature miR-29b increased 2000 and 2100 fold after treatment with TfNP29b but only 20 and 50 fold following treatment with NP29b in MV4-11 & Kasumi-1 respectively, compared to untreated or TfNPsc treated cells. Thus TfNPs were 40–100 times more efficient than NPs in increasing levels of miR-29b. Our observations were validated in primary blasts from 3 AML pts. At 48h miR-29b levels increase on average 400 fold in TfNP29b vs TfNPsc treated blasts. The levels of an unrelated miR (i.e. miR-140) remained unchanged in all our experiments. These results demonstrate that pM29b is efficiently delivered and processed into its mature form after TfNP delivery, leading to a specific increase of miR-29b in AML cell lines & primary blasts with otherwise low endogenous miR-29b expression. Downregulation of the miR-29b targets DNMT1, DNMT3A, and DNMT3B on RNA & protein level, determined by qPCR and western blotting respectively, was observed in both cell lines & primary AML samples at 48 hours following TfNP29b compared to TfNPsc treatment. The observed fold reduction on RNA level for DNMT1 was 0.63, 0.53 & 0.63, for DNMT3A 0.48, 0.53 & 0.58, and for DNMT3B 0.60, 0.73 & 0.53 in treated MV4-11, Kasumi-1 cells & pts blasts (mean, n=3), respectively. Increased miR-29b, following TfNP29b treatment resulted in antileukemic activity. Treatment with TfNP29b compared to TfNPsc led to 30% & 35% reduction in proliferation at day 5 and 50% & 64% decreased colony forming ability (scored after two weeks) in MV4-11 & Kasumi-1 cells, respectively. To investigate a possible sensitizing effect of miR-29b to DAC treatment, we treated MV4-11 cells and primary AML blasts with 2.5 uM DAC, which is comparable to concentrations achievable in treated AML pts. 72 hours after DAC treatment, decreased viability (measured by MTS assays) was observed in cells treated with TfNP29b (for 48h) followed by DAC compared with those treated with TfNPsc followed by DAC: 36% vs 53% (p

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.