The transcriptional regulator RovA positively regulates transcription of the Yersinia enterocolitica virulence gene inv. Invasin, encoded by inv, is important for establishment of Y. enterocolitica infection. However, a rovA mutant is more attenuated for virulence than an inv mutant, implying that RovA regulates additional virulence genes. When the Y. enterocolitica RovA regulon was defined by microarray analysis, YE1984 and YE1985 were among the genes identified as being upregulated by RovA. Since these genes are homologous to Xenorhabdus nematophila cytotoxin genes xaxA and xaxB, we named them yaxA and yaxB, respectively. In this work, we demonstrate the effects of YaxAB on the course of infection in the murine model. While a yaxAB mutant (⌬yaxAB) is capable of colonizing mice at the same level as the wild type, it slightly delays the course of infection and results in differing pathology in the spleen. Further, we found that yaxAB encode a probable cytotoxin capable of lysing mammalian cells, that both YaxA and YaxB are required for cytotoxic activity, and that the two proteins associate. YaxAB-mediated cell death occurs via osmotic lysis through the formation of distinct membrane pores. In silico tertiary structural analysis identified predicted structural homology between YaxA and proteins in pore-forming toxin complexes from Bacillus cereus (HBL-B) and Escherichia coli (HlyE). Thus, it appears that YaxAB function as virulence factors by inducing cell lysis through the formation of pores in the host cell membrane. This characterization of YaxAB supports the hypothesis that RovA regulates expression of multiple virulence factors in Y. enterocolitica.

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Background: Myc has proven extremely difficult to target therapeutically. Therefore, we hypothesized that optimal inhibition of several key targetable pathways involved in Myc signaling could overcome this long-standing problem. We identified phosphoinositide 3-kinase (PI3K), aurora kinase (Aurk), and bromodomain protein (BRD)4 as the primary therapeutic targets to counteract Myc deregulation based on strong evidence that these pathways are essential for tumor maintenance in Myc-driven malignancies. Methods: Cytotoxicity assays using MTS and trypan blue were used to compare levels of drug sensitivity in lymphoma cell lines with high and low Myc mRNA expression. Apoptosis and cell cycle assays were performed using Annexin V and Propidium Iodide staining. Murine xenograft models were used to assess the efficacy and tolerability of single vs. combined inhibition. Results: Myc-overexpressing Burkitt lymphoma (Raji) cells were treated with various concentrations of small molecule inhibitors against PI3K (BEZ-235), Aurk (MLN-8237), or BRD4 (I-BET-151) for 48 to 72 hours and cell viability was evaluated. BEZ-235, MLN-8237, and I-BET-151 inhibited cell growth individually with IC-50 of 30 nM, 10 nM, and 650 nM, respectively. Dual treatment with BEZ-235/MLN-8237, BEZ-235/I-BET-151, or MLN-8237/I-BET-151 induced more significant cell growth inhibition as compared to treatment with the single agent alone. The combination index (CI) values were less than 1 at various drug concentrations, indicating that different combinations of BEZ-235, MLN-8237, and I-BET-151 were synergistic in terms of inhibitory effect on tumor cell viability. Superior activity of the dual inhibition was also noted in other Myc-overexpressing lymphoma cells, including Ramos and SUDHL4. Combination treatments also increased apoptosis and induced more pronounced cell cycle arrest compared to the single agent treatment alone. We then analyzed protein expression by Western blot in Myc-overexpressing cells treated with various combinations of BEZ-235, MLN-8237, and I-BET-151. Treatment with BEZ-235 in Myc-overexpressing lymphoma cells resulted in reduced phosphorylated levels of the downstream effector RPS6K, which promotes protein translation and proliferation. MLN-8237 reduced the expression of p-HisH3 and p-Aurk while increasing the expression of p-S6K. Treatment with I-BET-151 resulted in significant reduction of Myc expression. Combined treatments had minimal impact on protein expression patterns compared to individual treatments, and the synergistic effect was independent of depletion of cytoplasmic levels of Myc. Lastly, athymic nude mice bearing Ramos lymphoma xenografts were treated with BEZ-235, MLN-8237, I-BET-151, and various dual-combinations of each agent. The mean tumor volumes in mice treated with negative control, BEZ-235, I-BET-151, and MLN-8237 as single agents were 3480, 2364, 2320, and 671 mm3, respectively, at Day 28. Mice treated with BEZ-235/I-BET-151, MLN-8237/I-BET-151, and BEZ-235/MLN-8237 combinations had mean tumor volumes of 1709, 461, and 166 mm3, respectively, at Day 28. The survival rates for mice treated with negative control, BEZ-235, I-BET-151, and MLN-8237 as single agents were 0%, 10%, 10%, and 70%, respectively, at Day 35. The combination of BEZ-235 and MLN-8237 was associated with significant toxicity with 60% of mice dying from weight loss and failure to thrive despite tumor regression. Mice treated with the MLN-8237 and I-BET-151 combination demonstrated the best survival rate of 100% at Day 35. Conclusion: Our data demonstrated that Myc-overexpressing tumors can be successfully targeted by inhibiting kinases associated with Myc-signaling. Specifically, MLN-8237, a small molecule inhibitor against AURK, induced apoptosis of Myc-overexpressing tumor cells in vitro and showed the most promising anti-tumor activity in mice bearing Myc-overexpressing lymphoma, especially when combined with I-BET-151, a BRD4 inhibitor. Citation Format: Steven I. Park, Carolina P. Lin, Michael Foote, Trevor Parton, David B. Darr, Daniel Roth, Aadra P. Bhatt, Dirk P. Dittmer, Norman E. Sharpless, Blossom Damania. Multitarget approach against PI3K, Aurora kinase, and BRD4 leads to improved antitumor activity in Myc-overexpressing lymphoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B39.

Histone post-translational modifications (PTMs) play a critical role in chromatin regulation. It has long been suspected that the poorly understood capability of ‘PTM-specific’ antibodies (i.e., their specificity and efficiency) is a prime driver of the reproducibility crisis in biomedical research. Here we confirm the validity of this concern as it applies to epigenomic mapping studies. Extensive spike-in panels of PTM-defined DNA-barcoded nucleosome standards show that >70% of >500 commercial antibodies (and >80% of the most highly cited) to >50 histone lysine methyl and acyl states have failing performance in ChIP (>20% cross reactivity/<5% target recovery; www.ChromatinAntibodies.com). Variable lot behavior (of both polyclonals and monoclonals) shows the danger of focusing on catalog numbers without considering the inherent variability of biological reagents. Ultimately, these studies support the inclusion of in situ standards to control genomic mapping assays as an improved path out of this morass. Despite these advances, the application of ChIP-seq for cancer research has been hampered by its requirement for high cell inputs and low reliability. The recent development of immunotethering assays, such as Cleavage Under Targets and Release Using Nuclease (CUT&RUN), deliver high signal-to-noise mapping data using a fraction of the required cells compared to ChIP-seq. These innovations enable the application of epigenomics for cancer research, particularly for primary cells, sorted populations, and clinical samples where cell numbers are limited. By adapting our nucleosome spike-in control approach for CUT&RUN assays, we show that while the same antibody problems observed in ChIP-seq also pervade CUT&RUN, continuous use of spike-in controls improves assay rigor and reproducibility to realize the potential of CUT&RUN to advance cancer research. Citation Format: Andrea L. Johnstone, Danielle N. Maryanski, Keli L. Rodriguez, Ellen N. Weinzapfel, Bryan J. Venters, Katherine Novitzky, Matthew R. Marunde, Carolina P. Lin, Michael-Christopher Keogh. A field survey of histone PTM antibodies in epigenomic mapping approaches reveals widespread liabilities: Best practices and resources for reliable epigenetic studies. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A022.

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Background: The proto-oncogene Myc is a key regulator of cell growth and survival, and aberrant Myc expression plays a significant role in various tumors, including non-Hodgkin lymphoma (NHL). Myc-associated lymphoma is clinically aggressive, more resistant to standard therapies, and associated with a significantly higher rate of mortality. Novel treatment paradigms are needed to improve survival of patients with Myc-associated NHL. Expression of Aurora Kinase (Aurk) has been associated with Myc, and Aurk is thought to be essential for the maintenance of Myc-driven lymphoma. Aurk is required for assembly of the mitotic spindle and plays key roles in cell proliferation. Amplification and overexpression of Aurk have been observed in various human tumors, including lymphoma, and are frequently associated with tumor progression as well as resistance to chemotherapy. Inhibition of Aurk may overcome resistance to chemotherapy and improve clinical outcomes in patients with Myc-overexpressing lymphoma. Methods: Cytotoxicity assays using MTS and trypan blue were used to compare levels of drug sensitivity in lymphoma cell lines resistant or sensitive to a conventional chemotherapeutic drug cyclophosphamide. Apoptosis and cell cycle assays were performed using Annexin V and Propidium Iodide staining. The Multiplexed Inhibitor Beads and quantitative Mass Spectrometry (MIB/MS) assays were used to profile kinome changes in response to Aurk inhibition. Murine xenograft models were used to assess the efficacy and tolerability of single vs. combined therapy. Results: Two Myc-overexpressing cell lines were identified as resistant (Raji) or sensitive (Ramos) to cyclophosphamide, with IC50 of ~ 400 µM and ~ 250 µM, respectively. Raji cells were characterized by increased expression of multidrug resistant protein 1 (MDR1) and mutated p53. There were no significant differences in baseline Aurk or Myc expressions between Raji and Ramos cells. Both cell lines were sensitive to alisertib, an aurora A kinase inhibitor, with maximum cytotoxicity achieved at ~ 100 nM. Combined treatment with alisertib and cyclophosphamide induced more significant cell growth inhibition as compared to treatment with the single agent alone. The combination index (CI) values were less than 1, indicating that alisertib was synergistic to cyclophosphamide in terms of inhibitory effect on tumor cell viability. Alisertib induced apoptosis and pronounced cell cycle arrest, resulting in polyploidy, in Raji cells. Alisertib had little to no effect on Myc, p53, or the total aurora A kinase protein expression in Raji cells although p-Histone-3-Ser10, a downstream target of Aurk, and p-Src levels were significantly decreased at 24 hours of treatment in vitro. Nocodazole-treated cells had reduced p-Aurk level and increased p-Rb as well as increased Mdm2 when treated with alisertib for 24 hours. Athymic nude mice bearing Ramos or Raji lymphoma xenografts were treated with cyclophosphamide, alisertib, or the combination. As expected, all mice bearing Ramos xenograft had complete tumor regression by day 35 of treatment while all mice bearing Raji xenograft had rapid disease progression with median survival of ~ 35 days when treated with cyclophosphamide alone. In contrast, when treated with the combination of cyclophosphamide and alisertib, all mice bearing Raji xenograft had complete regression of tumor by day 35 and had significant improvement in survival (median survival not reached by day 100) compared to the single agent control (p=0.022). Lastly, kinome analysis of Raji xenograft tumors treated with alisertib showed suppression of various kinases involved in Aurk, Src, and PI3K pathways. Western blot of the Raji tumors treated with a prolonged course (25 days) of alisertib showed significant decrease in p-Src and p53 protein levels. Conclusion: Our data demonstrates that alisertib induces synthetic lethality and overcomes chemoresistance in Myc-overexpressing tumors even in the presence of MDR1 overexpression and p53 mutation. The synergistic effect was largely independent of depletion of cytoplasmic level of Myc. Alisertib, when combined with a conventional chemotherapy drug, induced apoptosis and cell cycle arrest of Myc-overexpressing tumor cells in vitro and showed promising anti-tumor activity in mice bearing chemoresistant Myc-overexpressing lymphoma. Disclosures Park: Janssen: Other: travel; Seattle Genetics: Research Funding; Teva: Research Funding.