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Background Acute myeloid leukemia (AML) is an aggressive hematological cancer resulting from uncontrolled proliferation of differentiation-blocked myeloid cells. Seventy percent of AML patients are currently not cured with available treatments, highlighting the need of novel therapeutic strategies. A promising target in AML is the mammalian target of rapamycin complex 1 (mTORC1). Clinical inhibition of mTORC1 is limited by its reactivation through compensatory and regulatory feedback loops. Here, we developed a strategy to curtail these drawbacks through inhibition of an mTORC1 target, the eukaryotic initiation factor 4A (eIF4A). Methods We tested the anti-leukemic effect of a potent and specific eIF4A inhibitor (eIF4Ai), CR-1-31-B, in combination with cytosine arabinoside (araC) or the BCL2 inhibitor venetoclax. We utilized the MOLM-14 human AML cell line to model chemoresistant disease both in vitro and in vivo. In eIF4Ai-treated cells, we assessed for changes in survival and apoptosis, de novo protein synthesis, targeted intracellular metabolite content, bioenergetic profile, mitochondrial reactive oxygen species (mtROS) and mitochondrial membrane potential (MMP). Results eIF4i exhibits anti-leukemia activity in vivo while sparing non-malignant myeloid cells. In vitro, eIF4Ai synergizes with two therapeutic agents in AML, araC and venetoclax. eIF4Ai reduces mitochondrial membrane potential (MMP), the rate of ATP synthesis from mitochondrial respiration and glycolysis, and the expression of the antiapoptotic factors BCL2 and MCL1. Concomitantly, eIF4Ai decreases intracellular levels of specific metabolic intermediates of the tricarboxylic acid cycle (TCA cycle) and glucose metabolism, while enhancing mtROS. In vitro redox stress contributes to eIF4Ai cytotoxicity. Conclusions We discovered that AML cells rely on eIF4A-dependent cap translation for survival in vitro and in vivo. Our work indicates that eIF4A drives an intrinsic metabolic program sustaining bioenergetic and redox homeostasis. Furthermore, our work suggests that eIF4A-dependent cap translation contributes to adaptive processes involved in resistance to relevant therapeutic agents in AML.

Mass spectrometry (MS), particularly targeted proteomics, is increasingly being used for quantifying specific proteins and peptides in clinical specimens. The coupling of immuno-enrichment of proteotypic peptides with MS [e.g., immuno-multiple reaction monitoring (MRM) and immuno-matrix-assisted laser desorption ionization (MALDI)] enables the development of highly sensitive and specific assays for low-abundance signaling proteins. By incorporating stable isotope-labeled standards, these workflows allow the determination of endogenous protein concentrations. This is typically achieved through external calibration, often using surrogate matrices, which has inherent limitations for the analysis of clinical specimens as there are often substantial variations in the sample matrix, and sample amounts are typically limited. We have previously introduced the use of two peptide isotopologues for generating external calibration curves in plasma. Here, we present a two-point internal calibration (2-PIC) strategy using two isotopologues for immuno-MS assays and demonstrate its flexibility and robustness. Quantification of the tumor suppressor PTEN in Colo-205 cells by immuno-MRM and immuno-MALDI using 2-PIC and external calibration yielded very similar results (relative standard deviation between 2-PIC and external calibration: 4.9% for immuno-MRM; 1.1% for immuno-MALDI), without the need for a surrogate matrix or additional patient material for calibration, while concurrently reducing the instrument time and cost. Although our PTEN immuno-MRM and immuno-MALDI assays can be considered to be orthogonal as they utilized entirely different sample preparation and MS analysis workflows, targeted different PTEN peptides, and were performed in different laboratories, the endogenous Colo-205 PTEN levels determined with 2-PIC showed a good correlation (r2 = 0.9966) and good agreement (0.48 ± 0.01 and 0.29 ± 0.02 fmol/μg of total protein) between immuno-MRM and immuno-MALDI.

Chronic Lymphocytic Leukemia (CLL) is the most common adult leukemia in the western world. CLL consists of the accumulation of malignant B-cells in the blood stream and homing tissues. Although treatable, this disease is not curable, and resistance or relapse is often present. In many cancers, the study of metabolic reprograming has uncovered novel targets that are already being exploited in the clinic. However, CLL metabolism is still poorly understood. The ability of CLL lymphocytes to adapt to diverse microenvironments is accompanied by modifications in cell metabolism, revealing the challenge of targeting the CLL lymphocytes present in all different compartments. Despite this, the study of CLL metabolism led to an ongoing clinical trial using glucose uptake and mitochondrial respiration inhibitors. In contrast, glutamine and fatty acid metabolism remain to be further exploited in CLL. Here, we summarize the present knowledge of CLL metabolism, as well as the metabolic influence of Myc, ATM and p53 on CLL lymphocytes.

Buparlisib is an orally available pan-Class I PI3K inhibitor, that is more potent than idelalisib in vitro. Its distinct toxicities include hyperglycemia, hypertension, and mood disturbance. IND216...

PARP-3 is member of the PARP family of poly (ADP-ribose) polymerases involved in ADPribosylation. PARPs are involved in the basic mechanisms of DNA repair. PARP3, a critical player for efficient mitotic progression, is required for the stabilization of the mitotic spindle by regulation of the mitotic components, NuMA and Tankyrase 1. The sensitization effect of vinorelbine on PARP3 inhibition-induced cytotoxicity was assessed by the SRB assay. The contribution of programed cell death and cell cycle arrest to the sensitization effect were determined by assessing changes in Annexin V, a marker of apoptosis. Alterations in cell cycle progression were assessed by cell cycle analysis. We used immunofluorescence to assess the effect of vinorelbine and/or PARP3 inhibitors on tubulin and microtubule depolarization. The PARP3 chemiluminescent assay kit was used for PARP3 activity. PARP3 inhibitors sensitize breast cancer cells to vinorelbine, a vinca alkaloid used in the treatment of metastatic breast cancer. Olaparib which was originally described as a PARP1 and 2 inhibitor has recently been shown to be a potent PARP3 inhibitor while ME0328 is a more selective PARP3 inhibitor. The combination of vinorelbine with nontoxic concentrations of ME0328 or olaparib reduces vinorelbine resistance by 10 and 17 fold, respectively, potentiating vinorelbine-induced arrest at the G2/M boundary. In addition, PARP3 inhibition potentiates vinorelbine interaction with tubulin. Furthermore, olaparib or ME0328 potentiates vinorelbine-induced PARP3 inhibition, mitotic arrest, and apoptosis. Our results indicated this approach with PARP3 inhibitors and vinorelbine is unique and promising for breast cancer patients with metastases. This combination could significantly increase the survival of breast cancer patients with metastases.

e12561 Background: Ki-67 is a marker of proliferating cells. The recurrence score based on the 21-gene breast cancer assay also called Oncotype Dx provides prognostic and predictive information for recurrence in early stage breast cancer patients. We previously showed that there is a moderate correlation between Ki67 and oncotype Dx recurrence score. In this retrospective study, we aimed to examine whether high Ki67 could predict the distant recurrence in early stage breast cancer with low oncotype Dx scores ( < 25). Methods: This retrospective study included 278 consecutive cases of hormone receptor-positive, HER2 negative (T1-2 N0 M0) breast cancer who were diagnosed between 2008 and 2015 with low oncotype Dx ( < 25). Patients’ clinical outcome in terms of distant recurrence after breast surgery was determined up to December 2020 (median follow-up of 7 years). Patients were divided in to low risk (Ki67 < 15%) and high risk (Ki67 > = 15%) groups. Results: Of 278 cases with average and median age of 59 and 60 respectively, 148 (53%) were in Ki67 low risk and 130 (47%) were in Ki67 high risk group. Average and median oncotype Dx were 13.86 and 15 respectively in Ki67 low risk versus 15.23 and 16 respectively in Ki67 high risk group. 13 patients (4%) experienced distant metastasis in lung, liver, bone and skin. Of these 13 cases with average and median oncotype Dx 15.84 and 19 respectively, 12 (92%) were in the Ki67 high risk group and only 1 (8%) belonged to the low risk category. High Ki67 patients were overrepresented in group with recurrent distant metastasis compare to group without recurrent disease (Pearson Chi-Square = 51.18 with 1 degree of freedom and P = < 0.001). Conclusions: Ki67 high patients in the low risk oncotype Dx group are relapsing at a significantly higher rate suggesting that Ki67 combined with low oncotype Dx further refines the risk of distant relapse.

In this work we explored metabolic aspects of human primary leukemic lymphocytes that hold a potential impact on the treatment of Bruton tyrosine kinase (BTK)-driven diseases. Our results suggest that there is crosstalk between Bruton tyrosine kinase (BTK) signaling and bioenergetic stress responses. In primary chronic lymphocytic leukemia (CLL) lymphocytes, pharmacological interference with mitochondrial ATP synthesis or glucose metabolism affects BTK activity. Conversely, an inhibitor of BTK used clinically (ibrutinib) induces bioenergetic stress responses that in turn affect ibrutinib resistance. Although the detailed molecular mechanisms are still to be defined, our work shows for the first time that in primary B cells, metabolic stressors enhance BTK signaling and suggest that metabolic rewiring to hyperglycemia affects ibrutinib resistance in TP53 deficient chronic lymphocytic leukemia (CLL) lymphocytes.

Currently, there is no marker in use in the clinical management of colon cancer to predict which patients will respond efficiently to 5‐fluorouracil (5‐FU), a common component of all cytotoxic therapies. Our aim was to develop and validate a multigene signature associated with clinical outcome from 5‐FU therapy and to determine if it could be used to identify patients who might respond better to alternate treatments. Using a panel of 5‐FU resistant and sensitive colon cancer cell lines, we identified 103 differentially expressed genes providing us with a 5‐FU response signature. We refined this signature using a clinically relevant DNA microarray‐based dataset of 359 formalin‐fixed and paraffin‐embedded (FFPE) colon cancer samples. We then validated the final signature in an external independent DNA microarray‐based dataset of 316 stage III FFPE samples from the PETACC‐3 (Pan‐European Trails in Alimentary Tract Cancers) clinical trial. Finally, using a drug sensitivity database of 658 cell lines, we generated a list of drugs that could sensitize 5‐FU resistant patients using our signature. We confirmed using the PETACC‐3 dataset that the overall survival of subjects responding well to 5‐FU did not improve with the addition of irinotecan (FOLFIRI; two‐sided log‐rank test p = 0.795). Conversely, patients who responded poorly to 5‐FU based on our 12‐gene signature were associated with better survival on FOLFIRI therapy (one‐sided log‐rank test p = 0.039). This new multigene signature is readily applicable to FFPE samples and provides a new tool to help manage treatment in stage III colon cancer. It also provides the first evidence that a subgroup of colon cancer patients can respond better to FOLFIRI than 5‐FU treatment alone.

Poly (ADP-ribose) polymerase (PARP) resulting poly (ADP-ribosyl)ation is an early player in modification of proteins detected at single strand breaks and double strand breaks contributing to DNA repair. PARP1, 2 and 3 are involved in this process. However, PARP3 is unique in that, it is also required for efficient mitotic progression, specifically in stabilization of the mitotic spindle. The vinca alkaloids such as vinorelbine, arrest cells in metaphase due to inhibition of the assembly and dynamics of microtubules. Nontoxic concentrations of 2 PARP3 inhibitors, ME-0328 and olaparib, sensitized various her-2 negative and triple negative breast cancer cell lines greater than or equal to 10 fold to vinorelbine associated with potentiation of vinorelbine’s interaction with tubulin and also vinorelbine-induced PARP3 inhibition, mitotic arrest, and apoptosis without evidence of a DNA repair mechanism involved in this sensitization (Sharif-Askari B et al. Breast Cancer Res Treat. 2018 Nov;172(1):23-32). Eribulin is a Halichondrin B analogue, an antitubulin drug, with a unique mechanism of action; it inhibits microtubule dynamics via a novel mechanism of action which seems to involve binding to a unique site on tubulin resulting in the suppression of microtubule polymerization without affecting depolymerization along with sequestration of tubulin into nonfunctional aggregate. After impressive results in phase III trials (including one of the rare trials to show prolongation of survival in comparison to standard of care in heavily pretreated metastatic breast cancer patients) the U.S. Food and Drug Administration approved eribulin for treatment of patients with metastatic breast cancer. PARP3 inhibitors again sensitized at least 5 fold her-2 negative and triple negative breast cancer cell lines to eribulin. We will do similar studies with eribulin as we have done with vinorelbine including in-vivo studies. These studies could define a new therapy in the treatment of metastatic triple negative and ER+/her-2 negative breast cancer. The inhibition of PARP3 may increase the sensitivity of tumor cells to mitotic spindle poison chemotherapies. Our results should stimulate development of specific PARP3 inhibitors for clinical use. Citation Format: Bahram Sharif-Askari, Raquel Aloyz, Lawrence Panasci. A new combination therapy to treat metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 300.

Enhanced DNA damage repair is one mechanism involved in colon cancer drug resistance. Thus, targeting molecular components of repair pathways with specific small molecule inhibitors may improve the efficacy of chemotherapy. ABT-888 and VE-821, inhibitors of poly-ADP-ribose-polymerase (PARP) and the serine/threonine-kinase Ataxia telangiectasia related (ATR), respectively, were used to treat colon cancer cell lines in combination with the topoisomerase-I inhibitor irinotecan (SN38). Our findings show that each of these DNA repair inhibitors utilized alone at nontoxic single agent concentrations resulted in sensitization to SN38 producing a 1.4 to 3 fold reduction in the 50% inhibitory concentration (IC50) of SN38 in three colon cancer cell lines. When combined together, nontoxic concentrations of ABT-888 and VE-821 produced a 4.5 to 27 fold reduction in the IC50 of SN38 with the HCT-116 colon cancer cells demonstrating the highest sensitization as compared to LoVo and HT-29 colon cancer cells. Furthermore, the combination of all three agents was associated with maximal G2-M arrest and enhanced DNA-damage (γH2AX) in all three colon cancer cell lines. The mechanism of this enhanced sensitization was associated with: (a) maximal suppression of SN38 induced PARP activity in the presence of both inhibitors and (b) ABT-888 producing partial abrogation of the VE-821 enhancement of SN38 induced DNA-PK phosphorylation, resulting in more unrepaired DNA damage; these alterations were only present in the HCT-116 cells which have reduced levels of ATM. This novel combination of DNA repair inhibitors may be useful to enhance the activity of DNA damaging chemotherapies such as irinotecan and help produce sensitization to this drug in colon cancer.

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Enhanced DNA damage repair is one mechanism behind colon cancer drug resistance. Thus, targeting molecular components of repair pathways with specific small molecule inhibitors may improve the efficacy of chemotherapy. ABT-888 and VE-821, inhibitors of poly-ADP-ribose-polymerase (PARP) and the serine/threonine-kinase Ataxia telangiectasia related (ATR), respectively, were used to treat colon cancer cell lines in combination with the topoisomerase-I inhibitor irinotecan (SN38). Our findings show that each of these molecules at nontoxic single agent concentrations synergized with SN38 to produce a 2.2 to 3 fold reduction in the 50% inhibitory concentration (IC50) of SN38 (Table 1). When combined, nontoxic concentrations of ABT-888 and VE-821 produced a 4.5 to 27 fold reduction in the IC50 of SN38. Furthermore, the combination of all three agents was associated with maximal G2-M arrest and enhanced DNA-damage (γH2AX). The mechanism of this enhanced synergy was associated with maximal suppression of SN38 induced PARP activity in the presence of both inhibitors. Furthermore, VE-821 enhancement of SN38 induced DNA-PK phosphorylation was abrogated by ABT-888, resulting in more unrepaired DNA damage. This novel combination of DNA repair inhibitors may be useful to enhance the activity of DNA damaging chemotherapies such as irinotecan and help circumvent resistance to this drug in colon cancer. | Cell Line | VE-821 (μM) | ABT-888 (μM) | SN38 (IC50) ± SE (nM) | I value | P value | |:--------- | ----------- | ------------ | --------------------- | ------- | ------- | | LoVo | | | 13.5±1.2 | | | | | 1 | | 4.5±1.9 | 0.3 | 0.02 | | | 0.5 | | 5.8±2.0 | 0.4 | 0.03 | | | | 0.5 | 9.4±1.0 | 0.7 | 0.06 | | | 1 | 0.5 | 3.0±1.2 | 0.2 | 0.01 | | | 0.5 | 0.5 | 4.7±1.9 | 0.4 | 0.02 | | HCT-116 | | | 8.1±0.7 | | | | | 1 | | 3.5±0.5 | 0.5 | 0.01 | | | 0.5 | | 5.2±0.6 | 0.7 | 0.03 | | | | 0.5 | 3.9±1.1 | 0.5 | 0.03 | | | 1 | 0.5 | 0.3±0.03 | 0.1 | 0.01 | | | 0.5 | 0.5 | 2.1±0.4 | 0.3 | 0.01 | | HT-29 | | | 20.5±1.8 | | | | | 1 | | 9.3±0.6 | 0.6 | 0.01 | | | 0.5 | | 12.2±1.3 | 0.7 | 0.02 | | | | 0.5 | 12.9±0.7 | 0.6 | 0.02 | | | 1 | 0.5 | 3.9±0.5 | 0.3 | 0.01 | | | 0.5 | 0.5 | 6.1±0.2 | 0.4 | 0.01 | Table 1 Colon cancer cell lines LoVo, HCT-116 and HT-29 were treated with SN38 alone or in combination with various concentrations of the ATR inhibitor VE-821 and/or the PARP inhibitor ABT-888. The IC50 values of SN38 were significantly reduced in the presence of VE-821 and/or ABT-888 for all cell lines. Drug synergy (I) values were determined, according to Berenbaum (1978), by calculating the ratio: (IC50 SN38 with inhibitors/IC50 SN38 alone) + (inhibitor concentration used/IC50 inhibitor alone); I less than 1 = synergism, I equal to 1 = additive effect and I greater than 1 = antogonism. Citation Format: David Davidson, Atlal Abu-Sanad, Yunzhe Wang, Fatemeh Hasheminasab, Justin Panasci, Raquel Aloyz, Lawrence Panasci. Simultaneous inhibition of ATR and PARP greatly sensitizes colon cancer cell lines to irinotecan. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5327. doi:10.1158/1538-7445.AM2015-5327