Ramasamy Santhanam

MicroRNAs bind to Toll-like receptors to induce prometastatic inflammatory response

MicroRNAs (miRNAs) are small noncoding RNAs, 19–24 nucleotides in length, that regulate gene expression and are expressed aberrantly in most types of cancer. MiRNAs also have been detected in the blood of cancer patients and can serve as circulating biomarkers. It has been shown that secreted miRNAs within exosomes can be transferred from cell to cell and can regulate gene expression in the receiving cells by canonical binding to their target messenger RNAs. Here we show that tumor-secreted miR-21 and miR-29a also can function by another mechanism, by binding as ligands to receptors of the Toll-like receptor (TLR) family, murine TLR7 and human TLR8, in immune cells, triggering a TLR-mediated prometastatic inflammatory response that ultimately may lead to tumor growth and metastasis. Thus, by acting as paracrine agonists of TLRs, secreted miRNAs are key regulators of the tumor microenvironment. This mechanism of action of miRNAs is implicated in tumor–immune system communication and is important in tumor growth and spread, thus representing a possible target for cancer treatment.

miR-328 Functions as an RNA Decoy to Modulate hnRNP E2 Regulation of mRNA Translation in Leukemic Blasts

MicroRNAs and heterogeneous ribonucleoproteins (hnRNPs) are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner. Here, we report that loss of miR-328 occurs in blast crisis chronic myelogenous leukemia (CML-BC) in a BCR/ABL dose- and kinase-dependent manner through the MAPK-hnRNP E2 pathway. Restoration of miR-328 expression rescues differentiation and impairs survival of leukemic blasts by simultaneously interacting with the translational regulator poly(rC)-binding protein hnRNP E2 and with the mRNA encoding the survival factor PIM1, respectively. The interaction with hnRNP E2 is independent of the microRNAs seed sequence and it leads to release of CEBPA mRNA from hnRNP E2-mediated translational inhibition. Altogether, these data reveal the dual ability of a microRNA to control cell fate both through base pairing with mRNA targets and through a decoy activity that interferes with the function of regulatory proteins.

Downregulation of p53-inducible microRNAs 192, 194, and 215 impairs the p53/MDM2 autoregulatory loop in multiple myeloma development.

In multiple myeloma (MM), an incurable B cell neoplasm, mutation or deletion of p53 is rarely detected at diagnosis. Using small-molecule inhibitors of MDM2, we provide evidence that miR-192, 194, and 215, which are downregulated in a subset of newly diagnosed MMs, can be transcriptionally activated by p53 and then modulate MDM2 expression. Furthermore, ectopic re-expression of these miRNAs in MM cells increases the therapeutic action of MDM2 inhibitors in vitro and in vivo by enhancing their p53-activating effects. In addition, miR-192 and 215 target the IGF pathway, preventing enhanced migration of plasma cells into bone marrow. The results suggest that these miRNAs are positive regulators of p53 and that their downregulation plays a key role in MM development.

FTY720, a new alternative for treating blast crisis chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphocytic leukemia.

Blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome-positive (Ph1-positive) acute lymphocytic leukemia (ALL) are 2 fatal BCR/ABL-driven leukemias against which Abl kinase inhibitors fail to induce a long-term response. We recently reported that functional loss of protein phosphatase 2A (PP2A) activity is important for CML blastic transformation. We assessed the therapeutic potential of the PP2A activator FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride), an immunomodulator in Phase III trials for patients with multiple sclerosis or undergoing organ transplantation, in CML-BC and Ph1 ALL patient cells and in in vitro and in vivo models of these BCR/ABL+ leukemias. Our data indicate that FTY720 induces apoptosis and impairs clonogenicity of imatinib/dasatinib-sensitive and -resistant p210/p190(BCR/ABL) myeloid and lymphoid cell lines and CML-BC(CD34+) and Ph1 ALL(CD34+/CD19+) progenitors but not of normal CD34+ and CD34+/CD19+ bone marrow cells. Furthermore, pharmacologic doses of FTY720 remarkably suppress in vivo p210/p190(BCR/ABL)-driven [including p210/p190(BCR/ABL)(T315I)] leukemogenesis without exerting any toxicity. Altogether, these results highlight the therapeutic relevance of rescuing PP2A tumor suppressor activity in Ph1 leukemias and strongly support the introduction of the PP2A activator FTY720 in the treatment of CML-BC and Ph1 ALL patients.

Sp1/NFκB/HDAC/miR-29b Regulatory Network in KIT-Driven Myeloid Leukemia

The biologic and clinical significance of KIT overexpression that associates with KIT gain-of-function mutations occurring in subsets of acute myeloid leukemia (AML) (i.e., core binding factor AML) is unknown. Here, we show that KIT mutations lead to MYC-dependent miR-29b repression and increased levels of the miR-29b target Sp1 in KIT-driven leukemia. Sp1 enhances its own expression by participating in a NFkappaB/HDAC complex that further represses miR-29b transcription. Upregulated Sp1 then binds NFkappaB and transactivates KIT. Therefore, activated KIT ultimately induces its own transcription. Our results provide evidence that the mechanisms of Sp1/NFkappaB/HDAC/miR-29b-dependent KIT overexpression contribute to leukemia growth and can be successfully targeted by pharmacological disruption of the Sp1/NFkappaB/HDAC complex or synthetic miR-29b treatment in KIT-driven AML.

Preclinical activity of a novel CRM1 inhibitor in acute myeloid leukemia.

Chromosome maintenance protein 1 (CRM1) is a nuclear export receptor involved in the active transport of tumor suppressors (e.g., p53 and nucleophosmin) whose function is altered in cancer because of increased expression and overactive transport. Blocking CRM1-mediated nuclear export of such proteins is a novel therapeutic strategy to restore tumor suppressor function. Orally bioavailable selective inhibitors of nuclear export (SINE) that irreversibly bind to CRM1 and block the function of this protein have been recently developed. Here we investigated the antileukemic activity of KPT-SINE (KPT-185 and KPT-276) in vitro and in vivo in acute myeloid leukemia (AML). KPT-185 displayed potent antiproliferative properties at submicromolar concentrations (IC50 values; 100-500 nM), induced apoptosis (average 5-fold increase), cell-cycle arrest, and myeloid differentiation in AML cell lines and patient blasts. A strong down-regulation of the oncogene FLT3 after KPT treatment in both FLT3-ITD and wild-type cell lines was observed. Finally, using the FLT3-ITD-positive MV4-11 xenograft murine model, we show that treatment of mice with oral KPT-276 (analog of KPT-185 for in vivo studies) significantly prolongs survival of leukemic mice (P < .01). In summary, KPT-SINE are highly potent in vitro and in vivo in AML. The preclinical results reported here support clinical trials of KPT-SINE in AML.

PP2A-activating drugs selectively eradicate tki-resistant chronic myeloid leukemic stem cells

The success of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) depends on the requirement for BCR-ABL1 kinase activity in CML progenitors. However, CML quiescent HSCs are TKI resistant and represent a BCR-ABL1 kinase-independent disease reservoir. Here we have shown that persistence of leukemic HSCs in BM requires inhibition of the tumor suppressor protein phosphatase 2A (PP2A) and expression--but not activity--of the BCR-ABL1 oncogene. Examination of HSCs from CML patients and healthy individuals revealed that PP2A activity was suppressed in CML compared with normal HSCs. TKI-resistant CML quiescent HSCs showed increased levels of BCR-ABL1, but very low kinase activity. BCR-ABL1 expression, but not kinase function, was required for recruitment of JAK2, activation of a JAK2/β-catenin survival/self-renewal pathway, and inhibition of PP2A. PP2A-activating drugs (PADs) markedly reduced survival and self-renewal of CML quiescent HSCs, but not normal quiescent HSCs, through BCR-ABL1 kinase-independent and PP2A-mediated inhibition of JAK2 and β-catenin. This led to suppression of human leukemic, but not normal, HSC/progenitor survival in BM xenografts and interference with long-term maintenance of BCR-ABL1-positive HSCs in serial transplantation assays. Targeting the JAK2/PP2A/β-catenin network in quiescent HSCs with PADs (e.g., FTY720) has the potential to treat TKI-refractory CML and relieve lifelong patient dependence on TKIs.

Targeted Delivery of microRNA-29b by Transferrin-Conjugated Anionic Lipopolyplex Nanoparticles: A Novel Therapeutic Strategy in Acute Myeloid Leukemia

Purpose: miR-29b directly or indirectly targets genes involved in acute myeloid leukemia (AML), namely, DNMTs, CDK6, SP1, KIT, and FLT3. Higher miR-29b pretreatment expression is associated with improved response to decitabine and better outcome in AML. Thus, designing a strategy to increase miR-29b levels in AML blasts may be of therapeutic value. However, free synthetic miRs are easily degraded in bio-fluids and have limited cellular uptake. To overcome these limitations, we developed a novel transferrin-conjugated nanoparticle delivery system for synthetic miR-29b (Tf-NP-miR-29b). Experimental Design: Delivery efficiency was investigated by flow cytometry, confocal microscopy, and quantitative PCR. The expression of miR-29b targets was measured by immunoblotting. The antileukemic activity of Tf-NP-miR-29b was evaluated by measuring cell proliferation and colony formation ability and in a leukemia mouse model. Results: Tf-NP-miR-29b treatment resulted in more than 200-fold increase of mature miR-29b compared with free miR-29b and was approximately twice as efficient as treatment with non-transferrin–conjugated NP-miR-29b. Tf-NP-miR-29b treatment significantly downregulated DNMTs, CDK6, SP1, KIT, and FLT3 and decreased AML cell growth by 30% to 50% and impaired colony formation by approximately 50%. Mice engrafted with AML cells and then treated with Tf-NP-miR-29b had significantly longer survival compared with Tf-NP-scramble (P = 0.015) or free miR-29b (P = 0.003). Furthermore, priming AML cell with Tf-NP-miR-29b before treatment with decitabine resulted in marked decrease in cell viability in vitro and showed improved antileukemic activity compared with decitabine alone (P = 0.001) in vivo. Conclusions: Tf-NP effectively delivered functional miR-29b, resulting in target downregulation and antileukemic activity and warrants further investigation as a novel therapeutic approach in AML. Clin Cancer Res; 19(9); 2355–67. ©2013 AACR.

Clinical and pharmacodynamic activity of bortezomib and decitabine in acute myeloid leukemia

We recently reported promising clinical activity for a 10-day regimen of decitabine in older AML patients; high miR-29b expression associated with clinical response. Subsequent preclinical studies with bortezomib in AML cells have shown drug-induced miR-29b up-regulation, resulting in loss of transcriptional activation for several genes relevant to myeloid leukemogenesis, including DNA methyltransferases and receptor tyrosine kinases. Thus, a phase 1 trial of bortezomib and decitabine was developed. Nineteen poor-risk AML patients (median age 70 years; range, 32-84 years) enrolled. Induction with decitabine (20 mg/m(2) intravenously on days 1-10) plus bortezomib (escalated up to the target 1.3 mg/m(2) on days 5, 8, 12, and 15) was tolerable, but bortezomib-related neuropathy developed after repetitive cycles. Of previously untreated patients (age ≥ 65 years), 5 of 10 had CR (complete remission, n = 4) or incomplete CR (CRi, n = 1); 7 of 19 overall had CR/CRi. Pharmacodynamic analysis showed FLT3 down-regulation on day 26 of cycle 1 (P = .02). Additional mechanistic studies showed that FLT3 down-regulation was due to bortezomib-induced miR-29b up-regulation; this led to SP1 down-regulation and destruction of the SP1/NF-κB complex that transactivated FLT3. This study demonstrates the feasibility and preliminary clinical activity of decitabine plus bortezomib in AML and identifies FLT3 as a novel pharmacodynamic end point for future trials.

Ets1 and Ets2 are required for endothelial cell survival during embryonic angiogenesis.

The ras/Raf/Mek/Erk pathway plays a central role in coordinating endothelial cell activities during angiogenesis. Transcription factors Ets1 and Ets2 are targets of ras/Erk signaling pathways that have been implicated in endothelial cell function in vitro, but their precise role in vascular formation and function in vivo remains ill-defined. In this work, mutation of both Ets1 and Ets2 resulted in embryonic lethality at midgestation, with striking defects in vascular branching having been observed. The action of these factors was endothelial cell autonomous as demonstrated using Cre/loxP technology. Analysis of Ets1/Ets2 target genes in isolated embryonic endothelial cells demonstrated down-regulation of Mmp9, Bcl-X(L), and cIAP2 in double mutants versus controls, and chromatin immunoprecipitation revealed that both Ets1 and Ets2 were loaded at target promoters. Consistent with these observations, endothelial cell apoptosis was significantly increased both in vivo and in vitro when both Ets1 and Ets2 were mutated. These results establish essential and overlapping functions for Ets1 and Ets2 in coordinating endothelial cell functions with survival during embryonic angiogenesis.