It has been suggested that deficient protein trafficking to the cell membrane is the dominant mechanism associated with type 2 Long QT syndrome (LQT2) caused by Kv11.1 potassium channel missense mutations, and that for many mutations the trafficking defect can be corrected pharmacologically. However, this inference was based on expression of a small number of Kv11.1 mutations. We performed a comprehensive analysis of 167 LQT2-linked missense mutations in four Kv11.1 structural domains and found that deficient protein trafficking is the dominant mechanism for all domains except for the distal C-terminus. Also, most pore mutations—in contrast to intracellular domain mutations —were found to have severe dominant-negative effects when co-expressed with wild type subunits. Finally, pharmacological correction of the trafficking defect in homomeric mutant channels was possible for mutations within all structural domains. However, pharmacological correction is dramatically improved for pore mutants when co-expressed with wild type subunits to form heteromeric channels.

Short-term fasting protects mice from lethal doses of chemotherapy through undetermined mechanisms. Herein, we demonstrate that fasting preserves small intestinal (SI) architecture by maintaining SI stem cell viability and SI barrier function following exposure to highdose etoposide. Nearly all SI stem cells were lost in fed mice, whereas fasting promoted sufficient SI stem cell survival to preserve SI integrity after etoposide treatment. Lineage tracing demonstrated that multiple SI stem cell populations, marked by Lgr5, Bmi1, or HopX expression, contributed to fasting-induced survival. DNA repair and DNA damage response genes were elevated in SI stem/progenitor cells of fasted etoposide-treated mice, which importantly correlated with faster resolution of DNA double-strand breaks and less apoptosis. Thus, fasting preserved SI stem cell viability as well as SI architecture and barrier function suggesting that fasting may reduce host toxicity in patients undergoing dose intensive chemotherapy.stem cells | DNA damage | chemotherapy | fasting C ancer patients undergoing chemotherapy experience high rates of morbidity, despite regimens that attempt to balance timing and dose intensity to mitigate off-target effects and doselimiting toxicities (1-3). Interestingly, fasting has been shown to provide host-protective effects against high-dose chemotherapyinduced toxicity in preclinical and clinical studies. For example, etoposide, which forms a ternary complex with DNA and topoisomerase II causing DNA double-strand breaks (DSBs), is far less toxic if mice are fasted before treatment (4). Fasting has also been shown to protect normal, but not cancer cells, from the toxicity of chemotherapy, thereby extending the lifespan of tumorbearing mice (4-8).Because of the rapid rate of epithelial cell proliferation in the small intestine (SI), gastrointestinal (GI) toxicity is one of the most common complications for a variety of chemotherapeutic treatments (9). Therefore, we investigated if fasting was capable of mitigating the GI toxicity normally associated with high-dose chemotherapy. Herein, we demonstrate that mice allowed to feed ad libitum before receiving high-dose chemotherapy showed marked histological changes to SI epithelium before death. These histological changes reflected loss of regenerative capacity as a result of stem cell depletion as well as structural damage from inflammatory cell infiltrates, similar to the SI response to high-dose ionizing radiation (10). In contrast, SI homeostasis was preserved in fasted mice by protection of stem cell viability and prevention of proinflammatory cell infiltrates. These results indicate that fasting mitigates GI side effects associated with chemotherapy by activating pathways that preserve SI stem cell integrity and by maintaining barrier function.

SUMMARY The ARF and p53 tumor suppressors are thought to act in a linear pathway to prevent cellular transformation in response to various oncogenic signals. Here we show that loss of p53 function leads to an increase in ARF protein levels which function to limit the proliferation and tumorigenicity of p53-deficient cells by inhibiting an IFN-β-STAT1-ISG15 signaling axis. Human triple-negative breast cancer (TNBC) tumor samples with co-inactivation of p53 and ARF exhibit high expression of both STAT1 and ISG15, and TNBC cell lines are sensitive to STAT1 depletion. We propose that loss of p53 function and subsequent ARF induction creates a selective pressure to inactivate ARF, and propose that tumors harboring co-inactivation of ARF and p53 would benefit from therapies targeted against STAT1 and ISG15 activation.

Background: Dysregulated signaling through GPCRs is implicated in oncogenic RAS signaling in breast cancer (BC). The complex regulatory mechanisms that link RAS nodes can trigger an adaptive response (e.g. resistance) when a single RAS node is inhibited. Inhibition of multiple RAS nodes may thus be required to achieve durable antitumor responses. To identify patients with dysregulated RAS signaling tumors that may respond to RAS node inhibitors, an assay using an impedance biosensor was developed. The CELsignia RAS Activity Test measures GPCR-initiated signaling activity and PI3K, mTOR, and BCL's role in transducing this activity in live tumor cells. The current study set out to characterize the prevalence of dysregulated RAS signaling initiated through a GPCR in HER2-negative BC patients and the role played by PI3K, mTOR and BCL. Methods: Fresh tumor specimens from 69 HER2- BC patients were used to derive tumor cell samples. Dynamic live cell response to a GPCR agonist (LPA), a PI3K-α inhibitor (GDC-0077), a pan-PI3K inhibitor (copanlisib), a pan-PI3K/mTOR inhibitor (gedatolisib), and a BCL inhibitor (navitoclax) was measured using an xCELLigence impedance biosensor (Agilent Technologies). From these responses, the gross amount of GPCR-initiated signaling and corresponding participation of PI3K-α, all Class 1 PI3K-isoforms, mTORC1, and BCL was quantified and converted to a signaling score. Correlative analyses using FACS markers (RPS6, AKT, ERK, caspase 3) were performed. Results: Hyperactive RAS signaling (RASs+) initiated by GPCR pathways was found in 16 of 69 (23.1%; 95% CI=15%-34%) tumors. The signaling scores were bimodally distributed; for the 16 RASs+ and the 53 RASs- patient tumors, the mean scores were 486 (SD 248) and 91 (SD 73), respectively. For the 16 RASs+ tumors, inhibition of all Class 1 PI3K-isoforms attenuated, on average, 71% of the GPCR signaling, while inhibition of PI3K-α alone had only a nominal inhibitory effect. When mTOR inhibition was combined with pan-PI3K inhibition, GPCR signaling was further attenuated by 14%. The most complete attenuation of RAS-signaling occurred when PI3K, mTOR, and BCL were simultaneously inhibited. Early apoptosis markers (RPS6, AKT, ERK, caspase 3) were found most significantly in RASs+ tumors with pan-PI3K/mTOR inhibition and to a greater degree with pan-PI3K/mTOR/BCL inhibition. No apoptosis markers were found in RASs- tumors regardless of which RAS node was inhibited. Conclusions: These findings suggest that a significant sub-group of BC patients have a RAS-involved oncogenic driver that is responsive ex vivo to pan-PI3K/mTOR and pan-PI3K/mTOR/BCL inhibitors. A clinical trial to evaluate treatment response of this patient sub-group to combined PI3K/mTOR or PI3K/mTOR/BCL inhibitors is warranted. Citation Format: Salmaan Khan, Adrish Sen, Catherine Kuzmicki, Ian MacNeil, Aaron Broege, Sarah Mutka, Kelly Brass, Ky McCracken, Laura Milligan, Katja Kotke, Brian Sullivan, Lance Laing. Sub-group of HER2-negative breast cancer patients with hyperactive RAS network signaling identified: Dynamic pathway activity test identifies patients that may benefit from PI3K/mTOR or PI3K/mTOR/BCL inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 342.