Differential Control of Yersinia pestis Biofilm Formation In Vitro and in the Flea Vector by Two c-di-GMP Diguanylate Cyclases

Yersinia pestis forms a biofilm in the foregut of its flea vector that promotes transmission by flea bite. As in many bacteria, biofilm formation in Y. pestis is controlled by intracellular levels of the bacterial second messenger c-di-GMP. Two Y. pestis diguanylate cyclase (DGC) enzymes, encoded by hmsT and y3730, and one phosphodiesterase (PDE), encoded by hmsP, have been shown to control biofilm production in vitro via their opposing c-di-GMP synthesis and degradation activities, respectively. In this study, we provide further evidence that hmsT, hmsP, and y3730 are the only three genes involved in c-di-GMP metabolism in Y. pestis and evaluated the two DGCs for their comparative roles in biofilm formation in vitro and in the flea vector. As with HmsT, the DGC activity of Y3730 depended on a catalytic GGDEF domain, but the relative contribution of the two enzymes to the biofilm phenotype was influenced strongly by the environmental niche. Deletion of y3730 had a very minor effect on in vitro biofilm formation, but resulted in greatly reduced biofilm formation in the flea. In contrast, the predominant effect of hmsT was on in vitro biofilm formation. DGC activity was also required for the Hms-independent autoaggregation phenotype of Y. pestis, but was not required for virulence in a mouse model of bubonic plague. Our results confirm that only one PDE (HmsP) and two DGCs (HmsT and Y3730) control c-di-GMP levels in Y. pestis, indicate that hmsT and y3730 are regulated post-transcriptionally to differentially control biofilm formation in vitro and in the flea vector, and identify a second c-di-GMP-regulated phenotype in Y. pestis

Results from a phase I expansion cohort of the first-in-class oral HIF-2α inhibitor PT2385 in combination with nivolumab in patients with previously treated advanced RCC

558 Background: The transcription factor hypoxia-inducible factor (HIF)-2α has been established as an oncogenic driver in clear cell renal cell carcinoma (ccRCC) due to underlying VHL deficiency. Activation of HIF-2α can also promote immunosuppression. In preclinical models, HIF-2α inhibition demonstrated increased efficacy in combination with checkpoint inhibitors (Han et al. AACR 2016). In a Phase 1 dose escalation/expansion trial in heavily pre-treated advanced ccRCC patients, PT2385 monotherapy was associated with variability in drug exposure with higher therapeutic exposure associated with improved anti-tumor activity (Courtney et al. JCO 2018). Methods: In the current Phase 1 expansion cohort, patients with advanced ccRCC who had received 1-3 prior therapies (including at least one VEGF(R)-targeting agent) were treated with PT2385 (800 mg PO BID) in combination with nivolumab (3 mg/kg IV Q2Weeks) to evaluate safety, efficacy, and pharmacokinetics. Results: 50 patients were enrolled. Median age was 62 with 58% ECOG 1 and 42% ECOG 2. Median number of prior therapies was 1; 42% of patients received ≥2 prior lines of therapy. The most common all-grade AEs were anemia (46%), fatigue (46%), nausea (36%), and arthralgia (30%). The most common Grade 3 AE’s were anemia (4%), fatigue (4%), and hypoxia (4%). Two Grade 4 events of elevated ALT and increased lipase/amylase were observed. As of August 31, 2018, ORR = 22% (1 CR, 10 PR). At a median follow up of 12.4 months (m), median PFS was 7.3 m for all patients. Patients who had sub-therapeutic exposures ( < 300 ng/ml) of PT2385 (n = 17) had a median PFS of 4.7 m compared to patients with therapeutic exposures of PT2385 (n = 33), who had a median PFS of 10.0 m. Conclusions: The combination of PT2385 + nivolumab was well tolerated and demonstrated promising anti-tumor activity in advanced ccRCC patients, most notably in patients who achieved therapeutic exposure of PT2385. Single agent and combination studies with PT2977, a second-generation HIF-2α inhibitor with an improved PK profile, are ongoing. Clinical trial information: NCT02293980

Phase I/II study of the oral HIF-2 α inhibitor MK-6482 in patients with advanced clear cell renal cell carcinoma (RCC)

611 Background: Hypoxia-inducible factor (HIF)-2α is a transcription factor that is a key oncogenic driver in RCC. MK-6482 is a first-in-class small molecule HIF-2α inhibitor that blocks the heterodimerization of HIF-2α with HIF-1β and induces regression in mouse xenograft RCC models. Methods: Pts with advanced ccRCC who had received at least 1 prior therapy were enrolled in an expansion cohort from the first-in-human phase 1/2 study of MK-6482 in advanced solid tumors (NCT02974738). Pts were administered 120 mg of MK-6482 orally once daily. Primary end point: safety. Key secondary end points: ORR, duration of response (DOR), and PFS. Results: Fifty-five pts were enrolled in the dose expansion cohort. Median (range) number of prior therapies was 3 (1-9); 67% received anti–PD-1 and anti-VEGF agents. Five pts (9%) were favorable risk, 40 (73%) were intermediate risk, and 10 (18%) were poor risk by IMDC criteria. With a median follow-up of 13 mo the most common all-grade, all-cause AEs > 30% were anemia (75%), fatigue (67%), dyspnea (47%), nausea (33%), and cough (31%). Anemia (26%) and hypoxia (15%) were the most common grade 3 AEs. No grade 4/5 drug-related AEs were observed. ORR was 24% with 13 confirmed PRs. Thirty-one pts (56%) had SD, with a disease control rate (CR+PR+SD) of 80%. Thirty-five of 52 (67%) pts with baseline and postbaseline assessments had tumor shrinkage. Median DOR was not reached; 81% of pts had response ≥6 mo per Kaplan-Meier estimate. Sixteen pts (29%) continued treatment beyond 12 mo. By IMDC risk, 2/5 pts with favorable risk had PR (ORR = 40%), 10/40 with intermediate risk had PR (ORR = 25%), and 1/10 with poor risk had PR (ORR = 10%); disease control rate was 100%, 80%, and 70%, respectively. Median PFS for the total population was 11.0 mo; the 12 mo PFS rate was 49%. Median PFS for favorable, intermediate, and poor IMDC risk was 16.5, 11.0, and 6.9 mo, respectively. As of May 15, 2019, 30 pts (55%) discontinued due to PD, 2 (4%) due to AEs. Sixteen pts (29%) had treatment ongoing. Conclusions: MK-6482 is well tolerated with a favorable safety profile and demonstrated promising single-agent activity in heavily pretreated pts with ccRCC across IMDC risk groups. A phase 3 trial in a similar population is planned. Clinical trial information: NCT02974738