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Supplementary Methods and Legends from Increased AKT S473 phosphorylation after mTORC1 inhibition is rictor dependent and does not predict tumor cell response to PI3K/mTOR inhibition

Mammalian target of rapamycin (mTOR) regulates cellular processes important for progression of human cancer. RAD001 (everolimus), an mTORC1 (mTOR/raptor) inhibitor, has broad antitumor activity in preclinical models and cancer patients. Although most tumor lines are RAD001 sensitive, some are not. Selective mTORC1 inhibition can elicit increased AKT S473 phosphorylation, involving insulin receptor substrate 1, which is suggested to potentially attenuate effects on tumor cell proliferation and viability. Rictor may also play a role because rictor kinase complexes (including mTOR/rictor) regulate AKT S473 phosphorylation. The role of raptor and rictor in the in vitro response of human cancer cells to RAD001 was investigated. Using a large panel of cell lines representing different tumor histotypes, the basal phosphorylation of AKT S473 and some AKT substrates was found to correlate with the antiproliferative response to RAD001. In contrast, increased AKT S473 phosphorylation induced by RAD001 did not correlate. Similar increases in AKT phosphorylation occurred following raptor depletion using siRNA. Strikingly, rictor down-regulation attenuated AKT S473 phosphorylation induced by mTORC1 inhibition. Further analyses showed no relationship between modulation of AKT phosphorylation on S473 and T308 and AKT substrate phosphorylation patterns. Using a dual pan-class I phosphatidylinositol 3-kinase/mTOR catalytic inhibitor (NVP-BEZ235), currently in phase I trials, concomitant targeting of these kinases inhibited AKT S473 phosphorylation, eliciting more profound cellular responses than mTORC1 inhibition alone. However, reduced cell viability could not be predicted from biochemical or cellular responses to mTORC1 inhibitors. These data could have implications for the clinical application of phosphatidylinositol 3-kinase/mTOR inhibitors. [Mol Cancer Ther 2009;8(4):742–53]

Supplementary Figure 3 from Increased AKT S473 phosphorylation after mTORC1 inhibition is rictor dependent and does not predict tumor cell response to PI3K/mTOR inhibition

Supplementary Figure 1 from Increased AKT S473 phosphorylation after mTORC1 inhibition is rictor dependent and does not predict tumor cell response to PI3K/mTOR inhibition

Supplementary Figure 4 from Increased AKT S473 phosphorylation after mTORC1 inhibition is rictor dependent and does not predict tumor cell response to PI3K/mTOR inhibition

Supplementary Figure 2 from Increased AKT S473 phosphorylation after mTORC1 inhibition is rictor dependent and does not predict tumor cell response to PI3K/mTOR inhibition

Supplementary Figure 2 from The Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the ERα Pathway in Breast Cancer

Supplementary Figure 4 from The Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the ERα Pathway in Breast Cancer

Supplementary Figure 6 from The Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the ERα Pathway in Breast Cancer

Supplementary Figure 3 from The Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the ERα Pathway in Breast Cancer

A limited number of receptor tyrosine kinases (e.g., ErbB and fibroblast growth factor receptor families) have been genetically linked to breast cancer development. Here, we investigated the contribution of the Ret receptor tyrosine kinase to breast tumor biology. Ret was expressed in primary breast tumors and cell lines. In estrogen receptor (ER)α-positive MCF7 and T47D lines, the ligand (glial-derived neurotrophic factor) activated signaling pathways and increased anchorage-independent proliferation in a Ret-dependent manner, showing that Ret signaling is functional in breast tumor cells. Ret expression was induced by estrogens and Ret signaling enhanced estrogen-driven proliferation, highlighting the functional interaction of Ret and ER pathways. Furthermore, Ret was detected in primary cancers, and there were higher Ret levels in ERα-positive tumors. In summary, we showed that Ret is a novel proliferative pathway interacting with ER signaling in vitro. Expression of Ret in primary breast tumors suggests that Ret might be a novel therapeutic target in breast cancer. [Cancer Res 2008;68(10):3743–51]

Supplementary Figure 1 from The Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the ERα Pathway in Breast Cancer

Supplementary Figure 5 from The Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the ERα Pathway in Breast Cancer

Supplementary Legends for Figures 1-6, Table 1 and Methods and Materials from The Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the ERα Pathway in Breast Cancer

Supplementary Table 1 from The Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the ERα Pathway in Breast Cancer

Approximately 20% of all non-Hodgkin lymphoma (NHL) and 60% of indolent NHL (iNHL) cases are follicular lymphoma (FL),[1][1],[2][2] a disease considered treatable, but not curable, with currently available therapeutic options.[3][3] While survival in patients with FL has improved with the

Introduction: The approval of several new, targeted agents has been transformative in the treatment of CLL. Prospective clinical trial data support the use of Ven after Ibr in CLL (Jones JA et al. Lancet Oncol 2018); however, limited data are available on the inverse sequencing of these agents (Mato AR et al. Br J Haematol 2018; Anderson M et al. Blood 2017). Given the recent FDA approval of Ven + obinutuzumab in first-line (1L) CLL, an upsurge in Ibr-naïve pts needing therapy post-Ven is likely; characterizing this sequencing is of the upmost importance. Here we present a US multicentre, retrospective, chart-review analysis to explore outcomes of Ibr post-Ven, in Ibr-naïve pts with CLL. Methods: Efficacy and safety outcomes were investigated for pts with Ibr-naïve CLL, treated with Ven +/- CD20 monoclonal antibody (mAb), who developed progressive disease ([PD] or discontinued Ven) and received Ibr salvage therapy (+/- CD20 mAb). Analyses included pts in 1L or relapsed/refractory setting. Pts were treated between Feb 14, 2012 and Jun 6, 2019, across four institutions (US); data cutoff was Jul 18, 2019. Results: Data were available for 27 pts with CLL who received Ibr post-Ven - the largest cohort to date. Median age was 64 (41-79) years, median time from diagnosis to first therapy was 9.0 (0-117.7) months (mo), and the median number of therapies prior to Ven was 2 (0-9). Prior therapies were varied and included: 1 Bruton's tyrosine kinase inhibitor (BTKi; not Ibr), 3 lenalidomide, 1 pt received 9 lines of therapy (including: idelalisib, lenalidomide, anti-CD22 and temsirolimus), others received chemo- or chemoimmunotherapy, or CD20 mAb only. Median time from diagnosis to initiation of Ven was 56.3 (0-157.7) mo. At baseline, the median lymphocyte count was 2.2 (0.2-220.0) K/µL; 8/24 (33.3%) pts had a lymph node ≥ 5cm. All evaluable pts (26/26) had ≥1 unfavourable prognostic risk factor; 12/20 (60.0%) pts had del17p, 10/16 (62.5%) had del11q, 12/24 (50.0%) had complex karyotype (CK) and 13/15 (86.7%) pts had unmutated IGHV. A complete or partial response (CR or PR) to Ven was achieved in 4/26 (15.4%) and 18/26 (69.2%) evaluable pts, respectively. The median time to PD on Ven was 29.0 (1.0-118.0) mo, with a median treatment duration of 18.0 (0.1-64.3) mo. Pts discontinued Ven due to PD (n=18), consent withdrawal (n=2), non-compliance (n=1), and other (n=6; allogeneic stem cell transplantation n=2, physician decision n=3, not evaluable n=1). Prior to initiation of Ibr, the median lymphocyte count was 1.9 (0.01-179.0) K/µL; 15/26 (57.7%) pts had adenopathy, and 5/13 (38.5%) had a lymph node ≥ 5cm. Risk factors included: del17p (4/10; 40.0%), del11q (4/9; 44.4%), CK (8/17; 47.1%) and unmutated IGHV (11/14; 78.6%). Median time from Ven initiation to Ibr initiation was 31.9 (1.8-60.3) mo; median time to Ibr initiation post-Ven was 0.7 (0-39.7) mo. The overall response rate to Ibr was 56.0% (CR: 1/25, PR: 13/25). The time to progression on Ibr, post-Ven, varied from 3.0 to 53.0 mo (n=10). The median duration of Ibr therapy was 18.3 (3.7-53.2) mo and 20.0 (4.9-44.3) mo for those remaining on Ibr (8/27); the median follow-up time matched the median therapy duration. Nineteen pts discontinued Ibr due to: PD (n=9), physician decision (n=4), adverse events (AEs; n=2), transplant (n=2), symptomatic deterioration and unknown reason (n=1 each). The median number of therapies prior to Ven for the 9 pts who discontinued Ibr due to PD was 2 (1-9); 4/9 pts received novel targeted therapies. Richter's transformation occurred in 1 pt (1/9). The 2 pts who discontinued Ibr due to AEs experienced either atrial fibrillation (AF)/brain abscess or pneumonia after 11.6 and 18.2 mo of Ibr, respectively. Other notable AEs were: major bleeding (n=1), AF (n=2), infection (n=1), neutropenia (n=1), myalgia/arthralgia (n=2), and other cardiac event (n=1). Ibr dose reductions due to fatigue and general malaise occurred in 1 pt. Conclusions: With the limitations of a retrospective series using real-world data, these data suggest that Ibr had substantial clinical activity post-Ven in heavily pre-treated, high-risk CLL pts; no new safety signals arose. Larger, prospective studies are required to fully characterize the efficacy of Ibr after Ven. Meanwhile, salvage therapy with Ibr remains a good option for pts with CLL who relapse after Ven. Disclosures Brown: Sunesis: Consultancy; Pharmacyclics: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding; Loxo: Consultancy, Research Funding; Invectys: Other: Data safety monitoring board; Octapharma: Consultancy; Morphosys: Other: Data safety monitoring board; Janssen: Honoraria; Dynamo Therapeutics: Consultancy; Teva: Honoraria; Sun Pharmaceuticals: Research Funding; Genentech/Roche: Consultancy; Gilead: Consultancy, Research Funding; BeiGene: Consultancy; Catapult Therapeutics: Consultancy; AstraZeneca: Consultancy; Acerta Pharma: Consultancy; AbbVie: Consultancy; Juno/Celgene: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy, Research Funding. Davids:Research to Practice: Honoraria; AbbVie, Acerta Pharma, Adaptive, Biotechnologies, Astra-Zeneca, Genentech, Gilead Sciences, Janssen, Pharmacyclics, TG therapeutics: Membership on an entity's Board of Directors or advisory committees; AbbVie, Astra-Zeneca, Genentech, Janssen, MEI, Pharmacyclics, Syros Pharmaceuticals, Verastem: Consultancy; Acerta Pharma, Ascentage Pharma, Genentech, MEI pharma, Pharmacyclics, Surface Oncology, TG Therapeutics, Verastem: Research Funding. Chang:Genentech: Research Funding; Celgene: Research Funding; Adaptive Biotechnologies: Research Funding. Ma:Kite: Consultancy; Xeme: Research Funding; Abbvie: Research Funding; Beigene: Research Funding; Bioverativ: Consultancy; Incyte: Research Funding; Genentech: Consultancy; Astra Zeneca: Consultancy, Research Funding, Speakers Bureau; Gilead: Research Funding; Janssen: Consultancy, Speakers Bureau; Novartis: Research Funding; Juno: Research Funding; Acerta: Research Funding; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau. Biondo:Genentech, Inc.: Employment; F. Hoffmann-La Roche Ltd: Equity Ownership. Mun:Genentech: Employment, Equity Ownership. Breuleux:F. Hoffmann - La Roche Ltd: Employment, Equity Ownership; Gilead: Equity Ownership; Basilea Ltd: Equity Ownership. Wierda:Janssen: Research Funding; Xencor: Research Funding; Gilead Sciences: Research Funding; Pharmacyclics LLC: Research Funding; Cyclcel: Research Funding; Sunesis: Research Funding; AbbVie: Research Funding; KITE pharma: Research Funding; Miragen: Research Funding; Juno Therapeutics: Research Funding; GSK/Novartis: Research Funding; Oncternal Therapeutics Inc.: Research Funding; Loxo Oncology Inc.: Research Funding; Genentech: Research Funding; Acerta Pharma Inc: Research Funding.

Introduction: Undetectable minimal residual disease at Methods: MRD and PFS data from two phase 2 studies of venetoclax monotherapy in patients with R/R CLL were pooled: Study M14-032 (NCT02141282) of patients with R/R CLL who failed BCRi therapy and Study M13-982 (NCT01889186) of patients with R/R CLL (and 5 previously untreated) with del(17p). In both studies, venetoclax was administered at a final dose of 400mg/day after dose ramp-up until disease progression or prohibitive toxicity. Patients with informative PB or BM MRD data available were included in the analysis. MRD was evaluated by multi-color flow cytometry, with the majority of patients assessed at regional reference centers using assays based on the standardized method published by the European Research Initiative on CLL (ERIC) consortium (4-color in the EU; 6-color in Australia and North America). Quantitative MRD results were categorized as uMRD4, intermediate MRD, or high MRD (≥10-2). For the correlation analysis between PB and BM MRD, only patients with matched time points (same day) were included. The concordance of MRD between PB and BM was evaluated by linear correlation coefficient. PFS was evaluated using Kaplan-Meier estimates and hazard ratios between best PB MRD status, and estimated by the Cox proportional hazards regression. Results: Of the 285 patients from the two trials, 176 had informative MRD data in either PB or BM and were included in the analyses. For the PB and BM comparison, there were 42 samples with matched time points from 38 patients. PB MRD level correlated with BM MRD level (Figure 1; R2 = .96; P Conclusions: Venetoclax monotherapy induced high rates of PB uMRD4 and intermediate MRD in both CR and PR iwCLL categories. PB and BM MRD levels achieved with venetoclax were highly concordant. Achievement of either uMRD4 or intermediate MRD in PB with venetoclax was associated with longer PFS. Disclosures Wierda: AbbVie, Inc: Research Funding; Genentech: Research Funding. Roberts:Walter and Eliza Hall: Employment, Patents & Royalties: Employee of Walter and Eliza Hall Institute of Medical Research which receives milestone and royalty payments related to venetoclax; AbbVie: Research Funding; Genentech: Research Funding; Janssen: Research Funding. Ghia:AbbVie, Inc: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Sunesis: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; BeiGene: Honoraria, Research Funding. Brown:Janssen: Consultancy; Abbvie: Consultancy; Acerta / Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees; Sun Pharmaceutical Industries: Research Funding; Loxo: Consultancy; Beigene: Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy; Morphosys: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Consultancy; Invectys: Membership on an entity's Board of Directors or advisory committees; Sunesis: Consultancy; Roche/Genentech: Consultancy; Pharmacyclics: Consultancy; Boehringer: Consultancy; Celgene: Consultancy; Verastem: Consultancy, Research Funding; Gilead: Consultancy, Research Funding. Stilgenbauer:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Cymbalista:Gilead: Honoraria; Janssen: Honoraria; AbbVie, Inc: Honoraria; Sunesis: Research Funding. Lamanna:Verastem: Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; TG Therapeutics: Research Funding; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jannsen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Seymour:Celgene: Consultancy; AbbVie: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Research Funding. Böttcher:Celgene: Research Funding; Genentech: Research Funding; AbbVie, INc: Honoraria, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria. Breuleux:Roche: Employment, Equity Ownership; Gilead: Equity Ownership; Basilea: Patents & Royalties; Novartis: Patents & Royalties. Chyla:AbbVie, Inc: Employment, Equity Ownership. Zhou:AbbVie, Inc: Employment, Equity Ownership. Nielsen:AbbVie, Inc: Employment, Equity Ownership. Kim:Abbvie: Employment, Equity Ownership. Potluri:AbbVie: Employment, Equity Ownership. Maher:AbbVie, Inc: Employment, Equity Ownership. Hillmen:Alexion Pharmaceuticals, Inc: Consultancy, Honoraria; F. Hoffmann-La Roche Ltd: Research Funding; Novartis: Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences, Inc.: Honoraria, Research Funding.

Introduction: The management of CLL has been transformed in recent years by the introduction of a number of targeted agents including the BCL2 inhibitor VEN, and the Bruton's tyrosine kinase inhibitor IBR. In the US and EU, VEN is currently approved for patients who have received at least 1 prior therapy, whereas IBR has wider indications that include front-line therapy as well as R/R CLL. The mechanisms of action of VEN and IBR are complementary (Bose et al. F1000Research 2017); this has led to studies of combination therapy in patients with CLL (Wierda et al. J Clin Oncol Suppl. 2018). In addition, the documented activity of VEN in patients with IBR R/R disease (Jones et al. J Clin Oncol Suppl. 2016), together with current approvals, has resulted in VEN being suggested as a reasonable treatment choice for patients who discontinue IBR (Byrd et al. J Clin Oncol Suppl. 2018). However, the clinical benefit of IBR in patients pretreated with VEN is less clear. We present a post-hoc series, obtained after follow-up of 8 patients with R/R CLL who received IBR after fixed-duration VEN+rituximab (R) therapy in the MURANO randomized phase 3 study (NCT02005471) (Seymour et al. N Engl J Med 2018). Methods: In MURANO, 389 patients with R/R CLL were enrolled and treated with 6 cycles of VEN+R followed by VEN monotherapy once-daily for up to 2 years, or 6 cycles of bendamustine (B)+R. Progression-free survival (PFS), the primary study endpoint, was based on investigator assessment. The present case series focuses on VEN+R patients from MURANO who developed progressive disease (PD), and who subsequently received IBR. Eight patients in the VEN+R group with PD subsequently received IBR (Table 1). The number of lines of therapy prior to VEN+R ranged from 1 (n=7) to 4 (n=1; median 1). Prior to VEN+R therapy, 7 of the patients (87.5%) received fludarabine+cyclophosphamide+R (FCR); of these, 3 patients (42.8%) achieved a complete response (CR), 3 (42.8%) achieved a partial response (PR), and 1 patient (14.2%) had stable disease (SD). Of these 7 patients, 2 were considered refractory to FCR before VEN+R therapy. At baseline (before VEN+R), three of the patients (37.5%) had chromosome 17p-deletion, and two patients (25%) had baseline lymph nodes ≥5 cm- Four of the 8 patients had IBR dose interruption or modification due to neutropenia (n=2), clarithromycin treatment (n=1), or cutaneous nevus biopsy (n=1). Multiple skin abscesses were reported in 1 patient. One patient had atrial fibrillation, and 2 patients had arthralgia (1 had nearly resolved on follow-up), and there were no reports of major bleeding. Conclusions: In this series of patients with R/R CLL who received IBR following prior VEN+R in the MURANO study, IBR showed clinical activity and acceptable tolerability, with no new safety signals. Our results suggest that the use of IBR after relapse following VEN+R is a reasonable option in patients with CLL. More data will be collected from MURANO on patients progressing after VEN+R who are subsequently treated with IBR monotherapy. Disclosures Greil: Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Honoraria, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Other: TRAVEL, ACCOMMODATIONS, EXPENSES; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sandoz: Honoraria, Research Funding. Fraser:AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria. Leber:Novartis Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees. Marks:BMS: Honoraria; Servier: Honoraria; Merck: Honoraria. Middeke:Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees. Schary:AbbVie: Employment. Boyer:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Breuleux:Roche: Employment, Equity Ownership; Gilead: Equity Ownership; Basilea: Patents & Royalties; Novartis: Patents & Royalties. Crompton:Roche: Employment, Equity Ownership. Humphrey:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Marlton:Pfizer: Membership on an entity's Board of Directors or advisory committees; GlycoMimetics: Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

INTRODUCTION: Patients with chronic lymphocytic leukemia (CLL) have significantly decreased health related quality of life (HRQoL), particularly related to severe and progressive fatigue. Side effects of chemotherapies and the emotional burden of living with an often poor prognosis disease also negatively impact patient HRQoL. Venetoclax, an oral agent that targets the anti-apoptotic protein BCL2, has demonstrated high rates of deep and durable response in patients with relapsed/refractory (R/R) CLL, including those with 17p deletions, and has been shown to facilitate clinically relevant improvement in several key aspects of functioning and HRQoL. We evaluated the impact of venetoclax monotherapy on the quality of life of patients with R/R CLL. METHODS: VENICE II is an ongoing open-label, phase 3b, multicenter study (NCT02980731) that assessed patient-reported HRQoL in patients who were ≥18 years old with R/R CLL, including those with 17p deletion, TP53 mutations, and/or prior experience with B-cell receptor pathway inhibitor-containing (BCRi) therapy, treated with venetoclax monotherapy (5-week dose-titration, starting at 20mg once daily, then increased weekly to 50 mg, 100 mg, 200 mg, and 400 mg, followed by 400mg once daily). The primary endpoint was the mean change from baseline to Week 48 in the European Organization for Research and Treatment of Cancer Quality of Life Core Questionnaire (EORTC QLQ-C30) subscale. HRQoL subscales analyzed included: Global Health Status, Role Functioning, Emotional Functioning, Cognitive Functioning, Social Functioning, and Fatigue. The impact on QoL was also assessed on the CLL Module (EORTC QLQ-CLL16). Relevance of mean changes in HRQoL measures from baseline were analyzed based on minimum important difference (MID); a 5-10 point change was defined as MID, and >10 points was considered clinically meaningful.(Osoba, D., et al. J Clin Oncol. 1998;16:139-44. Osoba, D., et al. Qual Life Res. 1994;3:353-64.) Safety and adverse events (AEs) were also monitored. RESULTS: As of the data cutoff, April 30, 2018, the median time on study was 28 weeks (range: 1 - 73) and the median time on therapy was 23 weeks (range: 0.1 - 69) in this ongoing study. Of the 169 treated patients, 70% were male; the median age was 65 years (range: 24 - 86). Among those with available data, 17p deletions and TP53 mutations were confirmed in 34% (41/122) and 38% (19/50) of patients, respectively. Overall, 38%, 20%, and 42% of patients had one, two, and three (or more) prior lines of therapy respectively; 21% of patients had prior BCRi therapy. Clinically meaningful improvements from baseline were observed by week 12 and were sustained through week 48 in the EORTC-QLQ-C30 global health status and the role function, social function, and fatigue subscales (Table and Figure 1A) and EORTC-QLQ-CLL16 future health and disease effect subscales (Table and Figure 1B). Eighty-two percent of patients had at least 1 AE; most commonly observed AEs (≥10% of patients) were neutropenia (35%), diarrhea (17%), thrombocytopenia (15%), anemia (12%), nausea (12%), and upper respiratory infection (11%). Twenty-eight percent of patients had a serious AE, of which the most common were pneumonia (5%), febrile neutropenia (4%) and pyrexia (3%). Five percent of patients discontinued the study due to an AE. CONCLUSIONS: Preliminary data from this ongoing study suggest that patients with R/R CLL experienced improvement in several key aspects of functioning and quality of life with venetoclax monotherapy within the first 12 weeks which is sustained over time. Venetoclax monotherapy was well tolerated in R/R CLL patients. These findings are consistent with previous studies of R/R CLL patients who received venetoclax monotherapy. Disclosures Cochrane: Janssen: Membership on an entity's Board of Directors or advisory committees; Cilag: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Bristol-Myers Squibb: Honoraria; Calgene: Honoraria; Amgen: Honoraria; Novartis: Honoraria; MSD: Honoraria. Robak:AbbVie, Inc: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Gilead: Consultancy. Yeh:GNT Biotech & Medicals Crop.: Research Funding. Nikitin:AbbVie, Inc: Speakers Bureau. Breuleux:Roche: Employment, Equity Ownership; Gilead: Equity Ownership; Basilea: Patents & Royalties; Novartis: Patents & Royalties. Masud:AbbVie, Inc: Employment, Equity Ownership. Sail:AbbVie, Inc: Employment, Equity Ownership. Komlosi:AbbVie, Inc: Employment, Equity Ownership. Anderson:Walter and Eliza Hall: Employment, Patents & Royalties; AbbVie, Inc: Research Funding; Genentech: Research Funding.

A central sensor of the availability of growth factors, nutrients and energy sources, the mammalian target of rapamycin complex 1 (mTORC1) kinase plays a key role in tumor biology. Consequently, mTORC1 inhibitors have been shown to have broad antitumor activity pre-clinically in experimental tumor models as well as clinically in cancer patients. Strikingly, certain tumor types appear to be predisposed to respond to mTORC1 inhibition, a phenomenon related to deregulation of critical elements of the PI3K/mTORC1 pathway. In this review we address optimization of clinical development in the context of mTORC1 inhibitor-induced activation of survival pathways, crosstalk between different signaling modules involved in malignant transformation, definition of rational target combination scenarios and biologically based dosing and patient stratification strategies. Emphasis is given where possible to mTORC1 drug development decisions based on full clinical publications.

Adeno-associated virus type 2 (AAV2) provokes a DNA damage response that mimics a stalled replication fork. We have previously shown that this response is dependent on ataxia telangiectasia-mutated and Rad3-related kinase and involves recruitment of DNA repair proteins into foci associated with AAV2 DNA. Here, we investigated whether recombinant AAV2 (rAAV2) vectors are able to produce a similar response. Surprisingly, the results show that both single-stranded and double-stranded green fluorescent protein-expressing rAAV2 vectors are defective in producing such a response. We show that the DNA damage signaling initiated by AAV2 was not due to the virus-encoded Rep or viral capsid proteins. UV-inactivated AAV2 induced a response similar to that of untreated AAV2. This type of DNA damage response was not provoked by other DNA molecules, such as single-stranded bacteriophage M13 or plasmid DNAs. Rather, the results indicate that the ability of AAV2 to produce a DNA damage response can be attributed to the presence of cis -acting AAV2 DNA sequences, which are absent in rAAV2 vectors and could function as origins of replication creating stalled replication complexes. This hypothesis was tested by using a single-stranded rAAV2 vector containing the p5 AAV2 sequence that has previously been shown to enhance AAV2 replication. This vector was indeed able to trigger DNA damage signaling. These findings support the conclusion that efficient formation of AAV2 replication complexes is required for this AAV2-induced DNA damage response and provide an explanation for the poor response in rAAV2-infected cells.

Mammalian target of rapamycin (mTOR) regulates cellular processes important for progression of human cancer. RAD001 (everolimus), an mTORC1 (mTOR/raptor) inhibitor, has broad antitumor activity in preclinical models and cancer patients. Although most tumor lines are RAD001 sensitive, some are not. Selective mTORC1 inhibition can elicit increased AKT S473 phosphorylation, involving insulin receptor substrate 1, which is suggested to potentially attenuate effects on tumor cell proliferation and viability. Rictor may also play a role because rictor kinase complexes (including mTOR/rictor) regulate AKT S473 phosphorylation. The role of raptor and rictor in the in vitro response of human cancer cells to RAD001 was investigated. Using a large panel of cell lines representing different tumor histotypes, the basal phosphorylation of AKT S473 and some AKT substrates was found to correlate with the antiproliferative response to RAD001. In contrast, increased AKT S473 phosphorylation induced by RAD001 did not correlate. Similar increases in AKT phosphorylation occurred following raptor depletion using siRNA. Strikingly, rictor down-regulation attenuated AKT S473 phosphorylation induced by mTORC1 inhibition. Further analyses showed no relationship between modulation of AKT phosphorylation on S473 and T308 and AKT substrate phosphorylation patterns. Using a dual pan-class I phosphatidylinositol 3-kinase/mTOR catalytic inhibitor (NVP-BEZ235), currently in phase I trials, concomitant targeting of these kinases inhibited AKT S473 phosphorylation, eliciting more profound cellular responses than mTORC1 inhibition alone. However, reduced cell viability could not be predicted from biochemical or cellular responses to mTORC1 inhibitors. These data could have implications for the clinical application of phosphatidylinositol 3-kinase/mTOR inhibitors. [Mol Cancer Ther 2009;8(4):742–53]

A central sensor of the availability of growth factors, nutrients, and energy sources, the mammalian target of rapamycin complex 1 (mTORC1) is an intracellular serine/threonine kinase which plays a key role in mammalian cell growth, proliferation, survival, and metabolism. Inhibitors of the mTORC1 kinase, including rapamycin (sirolimus), RAD001 (everolimus), CCI-779 (temsirolimus), and AP23573 (deforolimus), have been shown to have broad antitumor activity pre-clinically in experimental tumor models as well as clinically in cancer patients. The central role of mTORC1 in cell growth and metabolism suggests a promising drug combination potential, with mTORC1 inhibition sensitizing tumor cells to other anticancer agents. In this chapter we review the pre-clinical data supporting the combination of targeted and cytotoxic therapeutics with mTORC1 inhibitors. We assess this in the context of molecular aspects of mTORC1 inhibition and cross talk between different signaling pathways involved in malignant transformation. The known role of mTORC1 in the development of resistance to cancer therapies, as well as the potential for bypass mechanisms which may contribute to resistance to mTORC1 inhibitors, is presented in the context of preventing resistance through rationale drug combinations. Where full manuscripts are available, reference to supporting clinical data is provided.

A limited number of receptor tyrosine kinases (e.g., ErbB and fibroblast growth factor receptor families) have been genetically linked to breast cancer development. Here, we investigated the contribution of the Ret receptor tyrosine kinase to breast tumor biology. Ret was expressed in primary breast tumors and cell lines. In estrogen receptor (ER)α-positive MCF7 and T47D lines, the ligand (glial-derived neurotrophic factor) activated signaling pathways and increased anchorage-independent proliferation in a Ret-dependent manner, showing that Ret signaling is functional in breast tumor cells. Ret expression was induced by estrogens and Ret signaling enhanced estrogen-driven proliferation, highlighting the functional interaction of Ret and ER pathways. Furthermore, Ret was detected in primary cancers, and there were higher Ret levels in ERα-positive tumors. In summary, we showed that Ret is a novel proliferative pathway interacting with ER signaling in vitro. Expression of Ret in primary breast tumors suggests that Ret might be a novel therapeutic target in breast cancer. [Cancer Res 2008;68(10):3743–51]

Heregulins (HRG) are known as soluble secreted growth factors that, on binding and activating ErbB3 and ErbB4 cell surface receptors, are involved in cell proliferation, metastasis, survival, and differentiation in normal and malignant tissues. Previous studies have shown that some HRG1 splice variants are translocated to the nucleus. By investigating the subcellular localization of HRGα1-241, nuclear translocation and accumulation in nuclear dot-like structures was shown in breast cancer cells. This subcellular distribution pattern depends on the presence of at least one of two nuclear localization sequences and on two domains on the HRG construct that were found to be necessary for nuclear dot formation. Focusing on the nuclear function of HRG, a mammary gland cDNA library was screened with the mature form of HRGα in a yeast two-hybrid system, and coimmunoprecipitation of endogenous HRG was done. The data reveal positive interactions of HRGα1-241 with nuclear factors implicated in different biological functions, including transcriptional control as exemplified by interaction with the transcriptional repressor histone deacetylase 2. In addition, HRGα1-241 showed transcriptional repression activity in a reporter gene assay. Furthermore, a potential of HRG proteins to form homodimers was reported and the HRG sequence responsible for dimerization was identified. These observations strongly support the notion that HRG1 splice variants have multifunctional properties, including previously unknown regulatory functions within the nucleus that are different from the activation of ErbB receptor signaling. (Mol Cancer Res 2006;4(1):27–37)

Background: BAL27862, a novel, synthetic, small molecule, is a potent inhibitor of tubulin polymerization that induces cancer cell death. BAL27862 elicits a unique microtubule (MT) phenotype, distinct from paclitaxel, vinblastine and colchicine, has broad in vitro anti-proliferative activity against a diverse range of human tumor lines (low nM IC50s) and induces significant antitumor responses in a range of animal models of human cancer when administered orally (p.o.) or intravenously (i.v.). In this study, BAL27862 activity in a panel of experimental breast cancer models was assessed.Materials and Methods: Anti-proliferative activity was analyzed using a monolayer (crystal violet) or soft agar (clonogenic) assay. Effects on MT phenotypes were assessed by immunofluorescence for α-tubulin. Efficacy was assessed in mouse xenograft models bearing chemosensitive and multidrug resistant human breast tumors.Results: The unique BAL27862-associated MT phenotype in interphase cells consisted of a partially collapsed MT network without peripheral MTs. In dividing cells, tiny MT asters were found scattered within the nuclear region. Potent anti-proliferative activity was demonstrated against 8 breast cancer cell lines (crystal violet assay IC50 range: 6.5 – 22 nM for the SKBR3, MCF7, BT474, T47D, BT549, MDA-MB231, MDA-MB453 and MDA-MB468 lines), although one line (HCC1937) appeared relatively insensitive (IC50: >1000 nM). Interestingly, two breast cancer lines were sensitive to BAL27862 treatment in a clonogenic assay (IC50/IC70: MAXF 401 = 13/18 nM; MAXF MX1 = 22/46 nM), despite one being relatively resistant to paclitaxel treatment (MAXF 401 = 11/48 nM; MAXF MX1 = 127/>3500 nM). Moreover, using monolayer assays, BAL27862 activity was retained against five tumor lines overexpressing the Pgp efflux pump (including MT-3/ADR mammary adenocarcinoma cells), which were up to several thousand-fold resistant to paclitaxel and vinblastine. BAL27862 showed little activity against human stem cells or peripheral blood mononucleocytes.When administered p.o or i.v. to mice at well tolerated doses, BAL27862 treatment elicited statistically significant antitumor activity (p≤0.05) in three chemosensitive human breast tumor xenograft models (including MAXF 401, MaCa 4049 and MT-3); resulting in a final %T/C (ratio of mean tumor volume of treated and control group x 100) equivalent to that observed with comparator cytotoxics using MTD schedules (e.g. final %T/C in MT-3 model: 36% BAL27862, 35% paclitaxel, 50% doxorubicin). Strikingly, significant antitumor activity was maintained in the Pgp-overexpressing MT-3/ADR xenograft model, where paclitaxel and doxorubicin were ineffective (final %T/C: 37% BAL27862, 112% paclitaxel, 108% doxorubicin).Conclusions: BAL27862 is a new tubulin-interacting agent with an apparently novel mechanism of action. A potent antitumor activity in experimental models of breast cancer, including chemorefractory models, strongly support further development of BAL27862 as a novel breast cancer treatment modality with a possibility for both i.v. and p.o. administration. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2093.

8529 Background: There is an unmet need for new treatment options in FL, particularly for heavily pretreated, high-risk patients refractory to anti-CD20 and chemotherapy. Idelalisib, a PI3Kδ inhibi...