Studies indicate that MAO-B is induced in peripheral inflammatory diseases. To target peripheral tissues using MAO-B inhibitors that do not permeate the blood-brain barrier (BBB) the MAO-B-selective inhibitor deprenyl was remodeled by replacing the terminal acetylene with a COH function, and incorporating a para-OCHAr motif (compounds 10a-s). Further, in compound 32 the C-2 side chain corresponded to CHCN. In vitro, 10c, 10j, 10k, and 32 were identified as potent reversible MAO-B inhibitors, and all four compounds were more stable than deprenyl in plasma, liver microsomal, and hepatocyte stability assays. In vivo, they demonstrated greater plasma bioavailability. Assessment of in vitro BBB permeability showed that compound 10k is a P-glycoprotein (P-gp) substrate and 10j displayed mild interaction. Importantly, compounds 10c, 10j, 10k, and 32 displayed significantly reduced BBB permeability after intravenous, subcutaneous, and oral administration. These polar MAO-B inhibitors are pertinent leads for evaluation of efficacy in noncentral nervous system (CNS) inflammatory disease models.

Background: Acute myeloid leukemia (AML) chimeric antigen receptor (CAR) T cell therapies are at early stages of testing in human clinical trials. We previously described the design of a CD33-specific dimerizing agent regulated immunoreceptor complex (DARIC33) that, in the presence of rapamycin (RAPA), switches from an "OFF" to "ON" state that activates T cells in response to tumor antigen. Here, we describe RAPA controlled activation and anti-AML activity of DARIC33 using human AML xenograft NSG mouse models and GMP-compliant T cell manufacturing methodologies. We find that low nanomolar whole blood concentrations of RAPA, below levels used for immunosuppression, are needed for DARIC33 to become active in vivo and exhibit potent CD33-specific anti-AML activity. Methods: Clinical scale, DARIC33 and control cell products were manufactured by Seattle Children's Therapeutics (SC-DARIC33) by stimulating equal numbers of CD4+ and CD8+ T cells with anti-CD3/CD28 microbeads in closed gas-permeable culture vessels followed by lentiviral vector transduction and expansion in serum-free media supplemented with IL7, IL15 and IL21. The activation and anti-AML activity of SC-DARIC33 assayed in vitro by measuring cytokine production or lysis of chromium labeled target cells in the presence or absence of RAPA. NSG mice inoculated with either 1x10 6 luciferase expressing MV4-11 (CD33+) or 0.5x10 6 Raji cells expressing a huCD33 transgene (Raji.CD33, CD19+/CD33+) were treated with SC-DARIC33 and RAPA. Concentrations of RAPA in mouse blood were quantified by LC-MS/MS (Charles River). Results: Donor matched SC-DARIC33 and control CD19 CAR T cell products exhibited similar surface markers of engraftment fitness (CD62L+CD45RA+) and capacity for anti-tumor (CD27+CCR7+) effector function. Following coculture of SC-DARIC33 and Raji.CD33 cells without RAPA, concentrations of IL-2, TNF-α or IFN-γ were not increased in comparison to Raji.CD33 cells cocultured with mock T cell products. However, when exposed to 1 nM RAPA and Raji.CD33 targets, SC-DARIC33 produced cytokines in quantities similar to (Fig A). To determine the concentration of RAPA required to activate SC-DARIC33 in patients, DARIC33 T cells, CD33+ AML targets, and graded concentrations of RAPA were added to allogeneic human whole blood samples and plasma was recovered after 24 hours of incubation. RAPA addition increased IFN-g release with an apparent EC50 = 2.6nM (Fig B). Anti-AML SC-DARIC33 activity and whole blood [RAPA WB] in tumor bearing mice were determined and compared to pediatric RAPA pharmacokinetic models to select appropriate clinical RAPA dose schedules. NSG mice inoculated with Raji.CD33 tumors treated with either 1x10 7 CD19 CAR T cells or 3x10 7 SC-DARIC33 T cells, followed by RAPA 0.1 mg/kg IP QOD, exhibited stringent control of tumor growth, demonstrating a 3:1 cell dose equivalency (Fig C). AML progression was also inhibited when NSG mice were inoculated with MV411 AML cells and subsequently treated with 10 7 SC-DARIC33 followed by 0.01 mg/kg RAPA QD (Fig D). Blood samples from tumor bearing mice obtained on day 15, 2 hours post RAPA administration, showed [RAPA WB] = 2.3 ± 1.3 ng/mL, overlapping with the in vitroand indicating very low concentrations of RAPA effectively modulate the "OFF-to-ON" state transition. Population PK models simulating various RAPA doses and schedules in pediatric patients found oral daily dosing of 0.5 mg/m 2 RAPA will achieve [RAPA WB] = 1-3 ng/mL in most patients (Fig E). Conclusion: Evaluation of GMP cell products and RAPA PK demonstrate that very low doses of RAPA are sufficient to regulate SC-DARIC33. To establish safety of SC-DARIC33 in humans, an upcoming phase 1 trial clinical trial evaluating SC-DARIC33 in pediatric AML patients will test escalating cell doses followed by low-dose RAPA administration from post T cell infusion days 2-21 using a Bayesian optimal interval (BOIN) design. Peripheral blood samples will be monitored for CD33+ myeloid cell recovery after cessation of RAPA dosing. These data will establish safety and support the feasibility of SC-DARIC33 CAR T cells to be reversibly modulated in an "OFF-ON-OFF" fashion by intermittent low-dose RAPA administration. Figure 1 Figure 1. Disclosures Price: bluebird, bio: Current Employment. Zhang: bluebird, bio: Current Employment. Sundaram: bluebird, bio: Current Employment. Lewis: bluebird, bio: Current Employment. Bilic: C4 Therapeutics: Current Employment. Xia: bluebird, bio: Current Employment. Krostag: bluebird, bio: Ended employment in the past 24 months. So: bluebird, bio: Current Employment. Martin: bluebird, bio: Current Employment. Leung: bluebird, bio: Ended employment in the past 24 months. Astrakhan: bluebird, bio: Current Employment. Pogson: bluebird, bio: Current Employment. Jarjour: bluebird, bio: Current Employment. Jensen: bluebird, bio: Research Funding.

Triple-negative breast cancers (TNBC) account for 15-25% of all breast cancers, have poor outcomes and high rates of relapse. Along with metastatic disease treatment options for TNBC are limited to that of conventional chemotherapy. The lack of targeted therapies for these cancers is a distinct unmet clinical need. YB-1, a transcription factor, is associated with 70% of TNBC and poor prognosis. While YB-1 is not easily druggable, p90 ribosomal S6 kinase (RSK), which lies upstream of YB-1 and activates it by phosphorylation of serine 102, is an ideal candidate. We recently reported that RSK inhibition decreases TNBC cell growth. Also RSK and YB-1 are implicated in invasion and therefore may play a role in metastatic spread. Herein, we asked whether RSK inhibitors would be beneficial for patients with metastatic disease. Our concern for treating metastatic disease was inspired by a study we conducted in 2222 patients with breast cancer. Women who had local, regional or distant metastases were at a much higher risk of dying. Remarkably those with distant metastases were 100 times for likely to die from breast cancer as compared to those without disseminated disease. Further, women with TNBC specifically had the worst outcomes and their time to death was the shortest of any breast cancer subtype. We therefore conducted a screen of 128 compounds, which are in clinical trials, in SUM149 TNBC cells and compared them to the RSK inhibitor BI-D1870. Most of these drugs failed to inhibit TNBC growth; however, BI-D1870 was highly active. These promising results point towards RSK as a potential molecular target for TNBC yet there are no inhibitors available for use in patients at this time. To further validate RSK as a target for TNBC we asked which of the four RSK isoforms (RSK1-4) are expressed. Only RSK 1 and 2 are expressed in breast cancer cell lines. Inhibiting RSK by siRNA or BI-D1870 suppressed the growth of TNBC cells by ~90%; however, there was less of an effect on non-TNBC cells where growth was attenuated by ~50%. RSK inhibition with siRNA also triggered apoptosis to a greater degree in TNBC cell lines. There was no growth inhibitory effect on normal mammary epithelial cells (184htrt). Further, RSK inhibition suppressed the growth of TNBC colonies in soft agar by ~90%. Kinexus antibody arrays were used to understand why loss of RSK suppressed the growth of TNBC. Of note, cell proliferation, invasion and apoptosis proteins were suppressed indicating the multifaceted benefit of inhibiting RSK. Next we asked whether the RSK/YB-1 pathway was active in metastases. We assessed activated RSK in a panel of metastatic murine breast cancer cell lines. The RSK pathway was more active in the metastatic cell lines compared to the non-metastatic cells. Taking this further, we compared the non-metastatic 67NR to the metastatic 4T1 cells where RSK 1 and 2 were more highly expressed in the latter. Likewise, P-YB-1S102 was more active. Transfecting 67NR cells with activated RSK1 increased their invasion. Conversely inhibiting RSK with BI-D1870 decreased the growth and invasion of the 4T1 cells. In a mouse xenograft model, P-YB-1S102 was highly expressed in the lung and liver metastases obtained from the 4T1 cells. Similarly, P-YB-1S102 was active in a distant metastases obtained from a patient with TNBC. Subsequently, we identified three RSK inhibitors from a chemical library screen. The most active agent, compound 2, had an in vitro IC50=10nM which was comparable to BI-D1870. Accordingly compound 2 inhibited TNBC growth and signaling through YB-1. In conclusion we identify RSK as a promising therapeutic target for TNBC that has the potential to suppress the growth of metastases. Citation Format: Anna L. Stratford, Rachel Berns, Pauline So, Mary R. Pambid, Fotovati Abbas, Samah Abu-Ali, Kaiji Hu, Kevin Bennewith, Edie Dullaghan, Sandra E. Dunn. The RSK/YB-1 pathway represents an opportunity for targeting TNBC and holds promise of treating metastases. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B065.