Vera Roginskaya

Involvement of Shc and Cbl-PI 3-kinase in Lyn-dependent proliferative signaling pathways for G-CSF

Granulocyte colony-stimulating factor (G-CSF) is the major hematopoietic factor which controls the production and differentiation of granulocytes. The G-CSF receptor (G-CSFR) belongs to the superfamily of the cytokine receptors, which transduce signals via the activation of cytosolic protein tyrosine kinases (PTK). To determine the role of specific PTK in G-CSF signaling we expressed the human G-CSFR in cell lines derived from DT40 B cells, which lack either the Src-related Lyn or Syk. Wild-type (wt) and syk-deficient cells underwent increased DNA synthesis in response to G-CSF; lyn-deficient cells did not. The purpose of these studies is to identify Lyns downstream effectors in mediating DNA synthesis. While G-CSF stimulated Ras activity in all cell lines, G-CSF failed to induce the tyrosine phosphorylation of Shc in lyn-deficient cells. G-CSF induced a statistically significant activation of Erk1/Erk2 Kinase or p90Rsk only in the wt cells. G-CSF induced the tyrosine phosphorylation of Cbl and increased activity of PI 3-kinase in wild-type and syk-deficient, but non in lyn-deficient, cells. Inhibition of Shc by over-expression of its SH2 or PTB domains or PI 3-kinase by either treatment with wortmannin or expression of the CblY731F mutant decreased G-CSF-induced DNA synthesis. Thus, the Lyn, Cbl-PI 3-kinase, and Shc/non-Ras-dependent pathways correlate with the ability of cells to respond to G-CSF with increased DNA synthesis.

Repression of c-Cbl leads to enhanced G-CSF Jak-STAT signaling without increased cell proliferation

Engagement of the Granulocyte-Colony-Stimulating Factor (G-CSF) receptor activates non-receptor protein tyrosine kinases Lyn and Jak2. We found that Lyn-deficient DT40 cells that express the G-CSF receptor (DT40GR) do not demonstrate G-CSF-induced mitogenic signaling. Lyn associates with and phosphorylates a small set of molecules, including c-Cbl. c-Cbl is an adaptor involved in cell growth and cytoskeletal reorganization, predominantly in hematopoietic cells. Using yeast two-hybrid analysis, we found that c-Cbl directly couples Lyn to PI 3-kinase. We also found that expression of the c-CblY731F mutant, which uncouples PI 3-kinase, resulted in the inhibition of G-CSF-induced proliferative signaling in DT40GR cells. As a complementary strategy, we sought to analyse the effects of c-Cbl deficiency in DT40GR cells. We isolated, cloned and sequenced the full-length cDNA for chicken c-Cbl and constructed antisense vectors. Antisense inhibition of c-Cbl expression in DT40GR cells led to enhanced Jak-STAT activation following G-CSF stimulation. Yet, this enhancement of Jak-STAT activation was associated with decreased G-CSF-induced PI 3-kinase activity and DNA synthesis. PI 3-kinase activity correlated with DNA synthesis and physiological levels of c-Cbl. Together, these data suggest that physiologic level of c-Cbl provides a growth stimulatory pathway for G-CSF and that enhanced Jak-STAT activation is not sufficient for G-CSF-induced growth.

Elevation of XPA protein level in testis tumor cells without increasing resistance to cisplatin or UV radiation

Most testicular germ cell tumors are curable using cisplatin‐based chemotherapy, and cell lines from these tumors are unusually sensitive to cisplatin and other DNA‐damaging agents. It has been suggested that this might be caused by a lower‐than normal nucleotide excision repair (NER) activity. Previous studies found that cell lines from testicular germ cell tumors have on average about one‐third the level of the NER protein XPA in comparison to cell lines from other tumors. We asked whether over‐expression of XPA protein would alleviate the cellular sensitivity and increase the DNA repair capacity of a testis tumor cell line. Increasing XPA levels in 833K cells by 10‐fold did not increase resistance to UV irradiation. XPA was localized to the cell nucleus in all cell lines, before and after exposure to UV‐radiation. 833K cells were proficient in removing UV radiation‐induced photoproducts from the genome and increased XPA did not enhance the rate of removal. Further, over‐expressing functional XPA protein did not correlate with increased resistance of 833K testis tumor cells to cisplatin. Thus, although the amount of XPA in this testis tumor cell line is lower than normal, it is sufficient for NER in vivo. The relative sensitivity of testis tumor cells to cisplatin, UV radiation, and other DNA damaging agents is likely related not to NER capacity, but to other factors such as the integrity of the p53 pathway in these cells.

The combination of thioxodihydroquinazolinones and platinum drugs reverses platinum resistance in tumor cells by inducing mitochondrial apoptosis independent of Bax and Bak

The effective management of tumors resistant to platinum drugs-based anticancer therapies is a critical challenge in current clinical practices. The proapoptotic Bcl-2 family proteins Bax and Bak are essential for cisplatin-induced apoptosis. Unfortunately, Bax and its related upstream endogenous apoptotic signaling pathways are often dysregulated in cancer cells. Strategies that are able to bypass Bax- and Bak-dependent apoptotic pathways will thus provide opportunities to overcome platinum drug resistance. We have identified the thioxodihydroquinazolinone mdivi-1 as a member of a novel class of small molecules that are able to induce Bax- and Bak-independent mitochondrial outer membrane permeabilization when combined with cisplatin, thereby efficiently triggering apoptosis in platinum-resistant tumor cells. In the present structure activity relationship (SAR) study of a computationally selected library of mdivi-1 related small molecules, we established a pharmacophore model that can lead to the enhancement of platinum drug efficacy and Bax/Bak-independent mitochondrial apoptosis. Specifically, we found that a thiourea function is necessary but not sufficient for the synergism of this class of thioxodihydroquinazolinones with cisplatin. We were also able to identify more potent mdivi-1 analogs through this SAR study, which will guide future designs with the goal to develop novel combination regimens for the treatment of platinum- and multidrug-resistant tumors.

Inhibiting mitochondrial DNA ligase IIIα activates caspase 1-dependent apoptosis in cancer cells

Elevated levels of DNA ligase IIIα (LigIIIα) have been identified as a biomarker of an alteration in DNA repair in cancer cells that confers hypersensitivity to a LigIIIα inhibitor, L67, in combination with a poly (ADP-ribose) polymerase inhibitor. Because LigIIIα functions in the nucleus and mitochondria, we examined the effect of L67 on these organelles. Here, we show that, although the DNA ligase inhibitor selectively targets mitochondria, cancer and nonmalignant cells respond differently to disruption of mitochondrial DNA metabolism. Inhibition of mitochondrial LigIIIα in cancer cells resulted in abnormal mitochondrial morphology, reduced levels of mitochondrial DNA, and increased levels of mitochondrially generated reactive oxygen species that caused nuclear DNA damage. In contrast, these effects did not occur in nonmalignant cells. Furthermore, inhibition of mitochondrial LigIIIα activated a caspase 1-dependent apoptotic pathway, which is known to be part of inflammatory responses induced by pathogenic microorganisms in cancer, but not nonmalignant cells. These results demonstrate that the disruption of mitochondrial DNA metabolism elicits different responses in nonmalignant and cancer cells and suggests that the abnormal response in cancer cells may be exploited in the development of novel therapeutic strategies that selectively target cancer cells. Cancer Res; 76(18); 5431-41. ©2016 AACR.

Dispersed single wall carbon nanotubes do not impact mitochondria structure or function, but technical issues during analysis could yield incorrect results

Mitochondria are the organelles of cells that generate a majority of the cells energy through ATP and are involved in programmed cell death through apoptosis. An understanding of non-specific targeting of nanomaterials, including single wall carbon nanotubes (SWCNTs), to organelles is important in trying to modulate cell function or determine the cellular toxicity with long term exposure. Here, we examine the impact of SWCNTs dispersed with Pluronic F127 and protein on mitochondria using a battery of standard tests. Seahorse XF24 analysis suggests complete loss of mitochondiral function, but this data is artifactual due to SWCNTs adsorbing onto the Seahorse probes. Imaging using the mitochondrial functional dye JC-1 gives inconclusive results owing to fluorescence quenching by SWCNTs. We observe no co-localization or reorganization of mitochondria in the presence of SWCNTs, although the results could have been misinterpreted had we not been correcting for significant fluorescence quenching by SWCNTs. In sum, the surface activity and fluorescence quenching of SWCNTs alter many traditional cellular assays. However, light emitting (luciferase) assays show that ATP levels are not altered with SWCNT treatment suggesting that mitochondiral function is not impacted as well as that light-emitting assays are an essential complimentary approach for quantitative, unambiguous cellular study of nanomaterials.

Evaluation of mitochondrial bioenergetics, dynamics, endoplasmic reticulum-mitochondria crosstalk, and reactive oxygen species in fibroblasts from patients with complex I deficiency

Mitochondrial complex I (CI) deficiency is the most frequent cause of oxidative phosphorylation (OXPHOS) disorders in humans. In order to benchmark the effects of CI deficiency on mitochondrial bioenergetics and dynamics, respiratory chain (RC) and endoplasmic reticulum (ER)-mitochondria communication, and superoxide production, fibroblasts from patients with mutations in the ND6, NDUFV1 or ACAD9 genes were analyzed. Fatty acid metabolism, basal and maximal respiration, mitochondrial membrane potential, and ATP levels were decreased. Changes in proteins involved in mitochondrial dynamics were detected in various combinations in each cell line, while variable changes in RC components were observed. ACAD9 deficient cells exhibited an increase in RC complex subunits and DDIT3, an ER stress marker. The level of proteins involved in ER-mitochondria communication was decreased in ND6 and ACAD9 deficient cells. |ΔΨ| and cell viability were further decreased in all cell lines. These findings suggest that disruption of mitochondrial bioenergetics and dynamics, ER-mitochondria crosstalk, and increased superoxide contribute to the pathophysiology in patients with ACAD9 deficiency. Furthermore, treatment of ACAD9 deficient cells with JP4-039, a novel mitochondria-targeted reactive oxygen species, electron and radical scavenger, decreased superoxide level and increased basal and maximal respiratory rate, identifying a potential therapeutic intervention opportunity in CI deficiency.

Flavopiridol-induced growth inhibition and apoptosis of myeloid and myeloma cell lines and blasts

Abstract Survival following the diagnosis of Acute Myeloid Leukemia (AML) or Multiple Myeloma (MM) is among the worst for patients with hematologic malignancies. Recent success with the tyrosine kinase inhibitor ST1571 in Chronic Myeloid Leukemia provides a proof of principle for signal transduction therapy and challenges us to find other kinase inhibitors. Flavopiridol is an inhibitor of cyclin-dependent kinases (CDKs), currently in phase II trials for a variety of malignancies. We have studied the effects of Flavopiridol on myeloid cell lines HL-60, 32Dc13, and TF-1 and on myeloma cell lines U-266 and RPMI-8226. The G150 at 48 hr for Flavopiridol was HL-60

Abstract 5344: Novel thioxodihydroquinazolinone small molecules for combination with platinum drugs to reverse platinum resistance through inducing mitochondrial apoptosis independent of Bax and Bak

Platinum drugs are among the most effective anticancer drugs against various types of cancers, and the effective treatment of tumors resistant to platinum drugs-based anticancer therapies (either intrinsic or acquired during treatment) is a critical challenge in the current clinic. Bax and Bak, which are the proapoptotic Bcl-2 family proteins, are essential for cisplatin-induced apoptosis. Unfortunately, Bax and its upstream apoptotic signaling pathways are often altered in cancer cells. Strategies that induce apoptosis bypassing Bax- and Bak-dependent pathway will thus provide new opportunities to overcome platinum drug resistance. We have identified the thioxodihydroquinazolinone, mdivi-1, as a member of a novel class of small molecules that when combined with cisplatin are able to induce Bax- and Bak-independent mitochondrial outer membrane permeabilization and subsequent apoptosis in platinum resistant tumor cells. In order to identify the pharmacophore that is minimally required for enhancing the efficacy of platinum drugs and bax/bak-independent mitochondrial apoptosis, we performed a structure activity relationship (SAR) study of a computationally selected library of mdivi-1 related small molecules through substructure and similarity searches in the small molecule library collection of the University of Pittsburgh Center for Chemical Methodologies and Library Development (UPCMLD). We were able to identify that a thiourea function is necessary but not sufficient for the synergism of this class of thioxodihydroquinazolinones with cisplatin. We have also identified more potent mdivi-1 analogs through this SAR study. These more potent novel thioxodihydroquinazolinone small molecules, when combined with cisplatin, induce a synergistic increase of replication stress and DNA damage, upregulation of a pro-apoptotic Bcl-2 family protein Noxa, and substantial mitochondrial dysfunction including mitochondrial uncoupling. Importantly, the combination of these thioxodihydroquinazolinones with cisplatin leads to efficient apoptosis in cells depleted of Bax and Bak. Taken together, we were able to establish a preliminary pharmacophore model of this class of thioxodihydroquinazolinones and identify more potent structures that are essential for guiding future compound optimization for preclinical studies. Given the essential roles of Bax and Bak in many different classes of chemotherapeutic and targeted therapeutic drugs, our thioxodihydroquinazolinone / platinum based combination approach provides a novel targeting strategy for a broad spectrum of drug resistant tumors. Citation Format: Wei Qian, Joseph Salamoun, Jingnan Wang, Vera Roginskaya, Bennett Van Houten, Peter Wipf. Novel thioxodihydroquinazolinone small molecules for combination with platinum drugs to reverse platinum resistance through inducing mitochondrial apoptosis independent of Bax and Bak. [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 5344. doi:10.1158/1538-7445.AM2015-5344

Abstract 1683: Novel combination approach using platinum drugs and mitochondrial division inhibitor 1 (mdivi-1) overcomes platinum resistance by synergistically inducing mitochondrial outer membrane permeabilization (MOMP) and apoptosis independent of Drp1

Platinum based anticancer agents, including cisplatin, are among the most effective and widely used anticancer drugs. However, inherent and acquired resistance to these drugs in tumors lead to treatment failure. We found a class of thioquinazolinone derivatives, including the mitochondrial division inhibitor-1 (mdivi-1), that overcome both the inherent and acquired resistance to cisplatin in tumor cell lines and also in primary ovarian tumors isolated from cisplatin refractory patients. The combination of cisplatin and mdivi-1 produces synergistic effect in inducing rapid apoptosis. Using Drp1 (a mitochondrial fission protein and the reported target of mdivi-1) knockout MEF cells we demonstrated that the combination of cisplatin and mdivi-1 overcomes cisplatin resistance in a Drp1-independent manner. Mdivi-1 causes replication defects and its combination with cisplatin triggers synergistic increases of replication stress and DNA damage. Comparing the treatment with cisplatin and mdivi-1 alone, the combination of the two agents enhances mitochondrial apoptotic signaling by specifically upregulating the expression of a pro-apoptotic Bcl-2 family protein Noxa, which promotes Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP). Intriguingly, the combination of cisplatin and mdivi-1 also causes mitochondrial swelling that triggers a Bax/Bak-independent MOMP. Thus, the combination of cisplatin and mdivi-1 activates two distinct mechanisms that converge on mitochondria contributing to the enhancement of MOMP. This novel combination represents a potential effective strategy in overcoming platinum drug resistance by combining cisplatin with compounds that together are able to trigger the coinitiation of Bax/Bak-dependent and-independent mitochondrial apoptotic signaling. (Work was supported by P30CA047904, UPCI and PA Dept of Health CURE program) Citation Format: Wei Qian, Jingnan Wang, Vera Roginskaya, Lee McDermott, Robert Edwards, Donna Stolz, Fabien Llambi, Douglas Green, Bennett Van Houten. Novel combination approach using platinum drugs and mitochondrial division inhibitor 1 (mdivi-1) overcomes platinum resistance by synergistically inducing mitochondrial outer membrane permeabilization (MOMP) and apoptosis independent of Drp1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1683. doi:10.1158/1538-7445.AM2014-1683