Abstract: The ErbB3‐binding protein 1 (Ebp1) has been reported as either an oncogenic regulator or a tumor suppressor in a variety of cancers. Here, we show that Ebp1 p48, a predominant expression isoform, is highly expressed in the majority of human colon tumor cells compared with normal adjacent tissues and its expression is required for the oncogenic activities of these cells. Depletion of Ebp1 expression in primary colon cancer cells inhibits cell proliferation, colony forming, and invasion in vitro as well as tumor formation in vivo and enhances cell sensitivity to irradiation. We further demonstrated that Ebp1 interacts with TIF‐90, a splice variant of transcription initiation factor IA (TIF‐IA) of the RNA polymerase I complex, allowing for regulation of ribosomal RNA (rRNA) synthesis and oncogenesis in human colon cancer cells. Moreover, Ebp1 expression is essential for Akt protected TIF‐90 stability by preventing TIF‐90's ubiquitination by Mdm2 and hence, its proteasomal degradation. The results of the present study support a mechanism of underlying oncogenic activities by means of Ebp1 through regulation of TIF‐90‐mediated rRNA synthesis and suggest the potential therapeutic treatment of colon cancer by targeting Ebp1 and its signaling.

Abstract: Transcription initiation factor 90 (TIF‐90), an alternatively spliced variant of TIF‐IA, differs by a 90 base pair deletion of exon 6. TIF‐90 has been shown to regulate ribosomal RNA (rRNA) synthesis by interacting with polymerase I (Pol I) during the initiation of ribosomal DNA (rDNA) transcription in the nucleolus. Recently, we showed that TIF‐90‐mediated rRNA synthesis can play an important role in driving tumorigenesis in human colon cancer cells. Here we show that TIF‐90 binds GTP at threonine 310, and that GTP binding is required for TIF‐90‐enhanced rRNA synthesis. Overexpression of activated AKT induces TIF‐90 T310, but not a GTP‐binding site (TIF‐90 T310N) mutant, to translocate into the nucleolus and increase rRNA synthesis. Complementing this result, treatment with mycophenolic acid (MPA), an inhibitor of GTP production, dissociates TIF‐90 from Pol I and hence abolishes AKT‐increased rRNA synthesis by way of TIF‐90 activation. Thus, TIF‐90 requires bound GTP to fulfill its function as an enhancer of rRNA synthesis. Both TIF variants are highly expressed in colon cancer cells, and depletion of TIF‐IA expression in these cells results in significant sensitivity to MPA‐inhibited rRNA synthesis and reduced cell proliferation. Finally, a combination of MPA and AZD8055 (an inhibitor of both AKT and mTOR) synergistically inhibits rRNA synthesis, in vivo tumor growth, and other oncogenic activities of primary human colon cancer cells, suggesting a potential avenue for the development of therapeutic treatments by targeting the regulation of rRNA synthesis by TIF proteins.

Abstract: ErbB3, a member of the epidermal growth factor receptor family, reportedly plays an essential role in the regulation of cancer progression and therapeutic resistance. Numerous studies have indicated that ErbB3 binding protein 1 (Ebp1), a binding partner for ErbB3, plays an important regulatory role in the expression and function of ErbB3, but there is no agreement as to whether Ebp1 also has an ErbB3‐independent function in cancer and how it might contribute to tumorigenesis. In this review, we will discuss the different functions of the two Ebp1 isoforms, p48 and p42, that may be responsible for the potentially dual role of Ebp1 in cancer growth.

Abstract: Induction of reactive oxygen species (ROS), an important process for the cytotoxicity of various acute myeloid leukemia (AML) therapies including hypomethylating agents (HMAs), concurrently activates the NF‐E2‐related factor 2 (Nrf2) antioxidant response pathway which in turn results in induction of antioxidant enzymes that neutralize ROS. In this study, we demonstrated that Nrf2 inhibition is an additional mechanism responsible for the marked antileukemic activity in AML seen with the combination of HMAs and venetoclax (ABT‐199). HMA and venetoclax combined treatment augmented mitochondrial ROS induction and apoptosis compared with treatment HMA alone. Treatment of AML cell lines as well as primary AML cells with venetoclax disrupted HMA decitabine‐increased nuclear translocation of Nrf2 and induction of downstream antioxidant enzymes including heme oxygenase‐1 and NADP‐quinone oxidoreductase‐1. Venetoclax treatment also leads to dissociation of B‐cell lymphoma 2 from the Nrf2/Keap‐1 complex and targets Nrf2 to ubiquitination and proteasomal degradation. Thus, our results here demonstrated an undiscovered mechanism underlying synergistic effect of decitabine and venetoclax in AML cells, elucidating for impressive results in antileukemic activity against AML in preclinical and early clinical studies by combined treatment of these drugs.

Abstract: BCL‐2 inhibition through venetoclax (VEN) targets acute myeloid leukemia (AML) blast cells and leukemic stem cells (LSCs). Although VEN-containing regimens yield 60–70% clinical response rates, the vast majority of patients inevitably suffer disease relapse, likely because of the persistence of drug-resistant LSCs. We previously reported preclinical activity of the ribonucleoside analog 8-chloro-adenosine (8-Cl-Ado) against AML blast cells and LSCs. Moreover, our ongoing phase I clinical trial of 8-Cl-Ado in patients with refractory/relapsed AML demonstrates encouraging clinical benefit. Of note, LSCs uniquely depend on amino acid-driven and/or fatty acid oxidation (FAO)-driven oxidative phosphorylation (OXPHOS) for survival. VEN inhibits OXPHOS in LSCs, which eventually may escape the antileukemic activity of this drug. FAO is activated in LSCs isolated from patients with relapsed AML. Methods Using AML cell lines and LSC-enriched blast cells from pre-treatment AML patients, we evaluated the effects of 8-Cl-Ado, VEN and the 8-Cl-Ado/VEN combination on fatty acid metabolism, glycolysis and OXPHOS using liquid scintillation counting, a Seahorse XF Analyzer and gene set enrichment analysis (GSEA). Western blotting was used to validate results from GSEA. HPLC was used to measure intracellular accumulation of 8-Cl-ATP, the cytotoxic metabolite of 8-Cl-Ado. To quantify drug synergy, we created combination index plots using CompuSyn software. The log-rank Kaplan–Meier survival test was used to compare the survival distributions of the different treatment groups in a xenograft mouse model of AML. Results We here report that VEN and 8-Cl-Ado synergistically inhibited in vitro growth of AML cells. Furthermore, immunodeficient mice engrafted with MV4-11-Luc AML cells and treated with the combination of VEN plus 8-Cl-Ado had a significantly longer survival than mice treated with either drugs alone ( p  ≤ 0.006). We show here that 8-Cl-Ado in the LSC-enriched population suppressed FAO by downregulating gene expression of proteins involved in this pathway and significantly inhibited the oxygen consumption rate (OCR), an indicator of OXPHOS. By combining 8-Cl-Ado with VEN, we observed complete inhibition of OCR, suggesting this drug combination cooperates in targeting OXPHOS and the metabolic homeostasis of AML cells. Conclusion Taken together, the results suggest that 8-Cl-Ado enhances the antileukemic activity of VEN and that this combination represents a promising therapeutic regimen for treatment of AML.

Abstract: MiR-126 and miR-155 are key microRNAs (miRNAs) that regulate, respectively, hematopoietic cell quiescence and proliferation. Herein we showed that in acute myeloid leukemia (AML), the biogenesis of these two miRNAs is interconnected through a network of regulatory loops driven by the FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD). In fact, FLT3-ITD induces the expression of miR-155 through a noncanonical mechanism of miRNA biogenesis that implicates cytoplasmic Drosha ribonuclease III (DROSHA). In turn, miR-155 down-regulates SH2-containing inositol phosphatase 1 (SHIP1), thereby increasing phosphor-protein kinase B (AKT) that in turn serine-phosphorylates, stabilizes, and activates Sprouty related EVH1 domain containing 1 (SPRED1). Activated SPRED1 inhibits the RAN/XPO5 complex and blocks the nucleus-to-cytoplasm transport of pre-miR-126, which cannot then complete the last steps of biogenesis. The net result is aberrantly low levels of mature miR-126 that allow quiescent leukemia blasts to be recruited into the cell cycle and proliferate. Thus, miR-126 down-regulation in proliferating AML blasts is downstream of FLT3-ITD–dependent miR-155 expression that initiates a complex circuit of concatenated regulatory feedback (i.e., miR-126/SPRED1, miR-155/human dead-box protein 3 [DDX3X]) and feed-forward (i.e., miR-155/SHIP1/AKT/miR-126) regulatory loops that eventually converge into an output signal for leukemic growth.

Abstract: Relapse of acute myeloid leukemia (AML) is attributed to the persistence of quiescent leukemia stem cells (LSCs). Bcl-2 inhibition has been shown to target primitive leukemia progenitors. Venetoclax (VEN) is a FDA-approved Bcl-2-selective inhibitor for the treatment of AML. Although the activity of single agent VEN in AML patients (pts.) is modest, clinical efficacy in newly diagnosed, older pts. unfit for intense chemotherapy has been shown when VEN is combined with the hypomethylating agents (HMAs) azacytidine and decitabine or with low-dose nucleoside analog cytarabine. We have recently shown that VEN in combination with HMAs augments oxidative stress in AML cells and provided a molecular mechanism for the VEN-HMA-regulated NF-E2-related factor 2 (Nrf2) antioxidant pathway that could explain the results observed in early clinical studies in AML. Although about 70% of pts. initially respond to these VEN treatment regimens, about 30% of pts. do not and diminished efficacy of VEN combination treatments have been observed in pts. harboring poor-prognosis markers such as FLT3-ITD. In addition, future relapse of a percentage of pts. treated with VEN combinations is expected. Thus, novel treatment options for are urgently needed. We previously reported that the ribose containing, RNA-directed nucleoside analog 8-chloro-adenosine (8-Cl-Ado) demonstrates cytotoxic activity against AML cells and LSCs in vitro and in vivo, without significantly affecting normal hematopoietic stem cells. Importantly, our initial, unpublished results from a phase I/II clinical trial with single agent 8-Cl-Ado in pts. with refractory/relapsed AML demonstrate encouraging clinical benefits. Moreover, we have reported that FLT3-ITD AML is particularly sensitive to 8-Cl-Ado, thus suggesting 8-Cl-Ado plus VEN as a potential novel therapeutic regimen for treatment of AML. We here report that the VEN plus 8-Cl-Ado combination inhibited in vitro growth and induced apoptosis in AML primary cells, LSCs and cell lines significantly more compared to treatment with the individual agents. For in vitro cell growth studies, combination indices of & lt;1 for all experimental and calculated drug concentrations demonstrated strong synergy between the two drugs in 2 human AML cell lines (MV4-11 and KG-1a) and in AML cells isolated from 2 pts. Moreover, immune compromised NSG mice engrafted with FLT3-ITD MV4-11 cells survived significantly longer when treated with VEN (20 mg·kg‒1·day‒1, daily oral) plus 8-Cl-Ado (50 mg·kg‒1·day‒1; osmotic pump), as compared to single agent or vehicle-treated mice (p & lt;0.006, VEN+8-Cl-Ado vs. 8-Cl-Ado; p & lt;0.001 VEN+8-Cl-Ado vs. VEN). LSCs depend on amino acid metabolism-driven and/or fatty acid oxidation (FAO)-driven oxidative phosphorylation (OXPHOS) for energy production. VEN is known to target LSCs through inhibition of OXPHOS by targeting amino acid uptake/metabolism. We report here that 8-Cl-Ado inhibited the FAO pathway and down-regulated the oxygen consumption rate (OCR), a marker for OXPHOS, in LSCs. However, whereas 500 nM of 8-Cl-Ado was sufficient to induce MV4-11 growth inhibition, 1 microM of 8-Cl-Ado was needed for maximum inhibitory effect on FAO. We also report that 8-Cl-Ado increased expression of the anti-apoptotic protein p53. It was previously reported that p53 induces FAO in LSCs. Knockdown of p53 by siRNA augmented the inhibitory effect of 8-Cl-Ado on FAO and OCR. Importantly, addition of VEN could completely overcome the p53-induced activation of FAO and OCR. Mechanistically, we show that 8-Cl-Ado inhibited ribosomal RNA (rRNA) synthesis, a prerequisite for cellular proliferation, through down-regulation of the transcription initiation factor 1 (TIF-IA) protein. Since TIF-1A negatively regulates p53 expression, the inhibition of TIF-1A by 8-Cl-Ado resulted in up-regulation of p53 and subsequent p53-induced upregulation of FAO and OCR, thus diminishing the suppressive effects of 8-Cl-Ado on FAO and OCR. We further show that the VEN plus 8-Cl-Ado combination strongly induced p53 signaling, as shown by activation and inhibition of downstream p21 and PCNA proteins, respectively. This combination also augmented DNA fragmentation and apoptosis in LSCs. Thus, our data suggest that the synergy seen in AML with the VEN plus 8-Cl-Ado combination can be explained at least in part due to augmented inhibition of FAO and OXPHOS and represents a promising novel treatment for AML. Disclosures Pullarkat: Dova: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Servier: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genetech: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie, Inc.: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Marcucci:Iaso Bio: Membership on an entity's Board of Directors or advisory committees; Abbvie: Speakers Bureau; Novartis: Speakers Bureau; Pfizer: Other: Research Support (Investigation Initiated Clinical Trial); Merck: Other: Research Support (Investigation Initiated Clinical Trial); Takeda: Other: Research Support (Investigation Initiated Clinical Trial). Rosen:Celgene: Speakers Bureau; NeoGenomics: Consultancy; Seattle Genetics: Consultancy; Aileron Therapeutics: Consultancy; Novartis: Consultancy; paradigm Medical Communications: Speakers Bureau; Abbvie: Speakers Bureau; Pebromene: Consultancy.

Abstract: AML patients (pts) carrying the fms-related tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) have a poor prognosis. Combination of chemotherapy with tyrosine kinase inhibitors (TKIs) has improved survival of these pts, but a large proportion of them still die of their disease. Nucleoside analogs (NAs) are the backbone of several upfront and salvage chemotherapy regimens for AML, including FLT3-ITD. Although these agents have significant antileukemic activity, they are not effective in eradicating leukemia stem cells (LSCs), the likely reason for treatment failures in AML. Consequently, new strategies are needed to improve the outcome for this and other molecular subsets of AML pts. 8-Cl-Ado is a novel, ribose-containing, RNA-directed nucleoside analog which, different from other NAs, is incorporated into newly transcribed RNA rather than in DNA, causing inhibition of RNA transcription. Among the AML molecular subsets, we identified FLT3-ITD blasts as one of the most sensitive to 8-Cl-Ado; however the mechanism of this differential effect remains unknown. Ex-vivo treatment with 8-Cl-Ado induced dose-dependent growth inhibition and apoptosis in FLT3-ITD AML cell lines and primary blasts including the LSC-enriched CD34+CD38- immunophenotypic subpopulation, with IC50s ranging from 200 nM to 1400 nM and a negligible effect on normal hematopoietic stem cells. In an orthotopic murine model, mice xenografted with FLT3-ITD-positive MV4-11 cells and treated (upon engraftment) with 75 mg 8-Cl-Ado/kg/day through an implanted osmotic pump survived significantly longer than vehicle-treated controls (median survival 45 days vs. 27.3 days, p=0.002). MicroRNA-155 (miR-155) is the most over-expressed miRNA in FLT3-ITD and reportedly plays a key role in FLT3-ITD blast hyper-proliferation [PMID 20656931, PMID 25971362]. Thus, silencing of miR-155 has been proposed as a novel therapeutic approach for FLT3-ITD AML [PMID 25971362] . As 8-Cl-Ado is incorporated mainly into RNA, we reasoned that it could also be incorporated into miR-155 (and other miRNAs). Consistent with our hypothesis, we detected co-localization of 8-Cl-Ado and miR-155 in FLT3-ITD primary blast cells and MV4-11, using fluorescence-labeled 8-Cl-Ado (8-Cl-Ado-FAM) and miR-155 staining (SmartFlare probes), suggesting that 8-Cl-Ado interacts directly with miR-155. Using quantitative RT-PCR we demonstrated ~50% miR-155 down-regulation in 8-Cl-Ado-FAM or 8-Cl-Ado-treated MV4-11 cells and FLT3-ITD primary blast cells, compared to vehicle-treated controls. On a molecular level, 8-Cl-Ado-dependent miR-155 down-regulation was accompanied by up-regulation of SHIP1, a bona fide miR-155 target phosphatase that decreased p-AKT levels thereby negatively regulating FLT3-ITD-dependent AKT signaling required for leukemia cell growth and survival. This effect also disrupted the interaction of AKT and ErbB3 binding protein (Ebp1), a highly expressed protein that regulates p53 expression and prevents DNA fragmentation and apoptosis in normal and leukemic cells. Thus, we hypothesized by disrupting the AKT/Ebp1 interplay via miR-155 down-regulation, 8-Cl-Ado induces pro-apoptotic Ebp1-dependent p53 expression and activation and leukemia cell death. Indeed, we showed that 8-Cl-Ado treatment caused AKT/Ebp1 dissociation and p53 activation in primary FLT3-ITD AML blasts. Conversely, overexpression of miR-155 reversed 8-Cl-Ado-induced apoptosis. By combining 8-Cl-Ado with the TKI quizartinib we elicited a synergistic anti-leukemic effect in primary AML blasts [combination index (CI) values 〈 1 at doses required to inhibit 50, 75, and 90% of cell growth (ED50, ED75, ED90)]. In an orthotopic mouse model where FLT3-ITD-positive MV4-11 cells were xenografted, mice treated with 75 mg 8-Cl-Ado/kg/day plus quizartinib (0.5 mg/kg/day) survived significantly longer than mice treated with the individual agents or vehicle alone (median survival 54.2 days vs. 27.3 days for controls, p=0.016). Taken together, these results support 8-Cl-Ado as a promising novel agent via a unique mechanism of action for FLT3-ITD AML, mediated by miR-155 degradation. 8-Cl-Ado also synergizes with TKIs both in vitro and in vivo thereby representing a potentially novel clinical approach for FLT3-ITD AML. A single-agent phase I/II clinical trial in relapsed/refractory AML is already ongoing at our institution. Figure. Figure. Disclosures Wennerberg: Novartis: Research Funding. Gandhi:Pharmacyclics: Research Funding.

Abstract: The mechanisms underlying the transformation of chronic myeloid leukemia (CML) from chronic phase (CP) to blast crisis (BC) are not fully elucidated. Here, we show lower levels of miR-142 in CD34 + CD38 − blasts from BC CML patients than in those from CP CML patients, suggesting that miR-142 deficit is implicated in BC evolution. Thus, we create miR-142 knockout CML (i.e., miR-142 −/− BCR-ABL ) mice, which develop BC and die sooner than miR-142 wt CML (i.e., miR-142 +/+ BCR-ABL ) mice, which instead remain in CP CML. Leukemic stem cells (LSCs) from miR-142 −/− BCR-ABL mice recapitulate the BC phenotype in congenic recipients, supporting LSC transformation by miR-142 deficit. State-transition and mutual information analyses of “bulk” and single cell RNA-seq data, metabolomic profiling and functional metabolic assays identify enhanced fatty acid β-oxidation, oxidative phosphorylation and mitochondrial fusion in LSCs as key steps in miR-142-driven BC evolution. A synthetic CpG-miR-142 mimic oligodeoxynucleotide rescues the BC phenotype in miR-142 −/− BCR-ABL mice and patient-derived xenografts.