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Purpose: The phase II CAPTIVATE study investigated first-line treatment with ibrutinib plus venetoclax for chronic lymphocytic leukemia in two cohorts: minimal residual disease (MRD)-guided randomized treatment discontinuation (MRD cohort) and fixed duration (FD cohort). We report tumor debulking and tumor lysis syndrome (TLS) risk category reduction with three cycles of single-agent ibrutinib lead-in before initiation of venetoclax using pooled data from the MRD and FD cohorts. Patients and Methods: In both cohorts, patients initially received three cycles of ibrutinib 420 mg/day then 12 cycles of ibrutinib plus venetoclax (5-week ramp-up to 400 mg/day). Results: In the total population (N = 323), the following decreases from baseline to after ibrutinib lead-in were observed: percentage of patients with a lymph node diameter ≥5 cm decreased from 31% to 4%, with absolute lymphocyte count ≥25 × 109/L from 76% to 65%, with high tumor burden category for TLS risk from 23% to 2%, and with an indication for hospitalization (high TLS risk, or medium TLS risk and creatinine clearance Conclusions: Three cycles of ibrutinib lead-in before venetoclax initiation provides effective tumor debulking, decreases the TLS risk category and reduces the need for hospitalization for intensive monitoring for TLS.

Purpose:The phase II CAPTIVATE study investigated first-line treatment with ibrutinib plus venetoclax for chronic lymphocytic leukemia in two cohorts: minimal residual disease (MRD)-guided randomized treatment discontinuation (MRD cohort) and fixed duration (FD cohort). We report tumor debulking and tumor lysis syndrome (TLS) risk category reduction with three cycles of single-agent ibrutinib lead-in before initiation of venetoclax using pooled data from the MRD and FD cohorts.Patients and Methods:In both cohorts, patients initially received three cycles of ibrutinib 420 mg/day then 12 cycles of ibrutinib plus venetoclax (5-week ramp-up to 400 mg/day).Results:In the total population (N = 323), the following decreases from baseline to after ibrutinib lead-in were observed: percentage of patients with a lymph node diameter ≥5 cm decreased from 31% to 4%, with absolute lymphocyte count ≥25 × 109/L from 76% to 65%, with high tumor burden category for TLS risk from 23% to 2%, and with an indication for hospitalization (high TLS risk, or medium TLS risk and creatinine clearance Conclusions:Three cycles of ibrutinib lead-in before venetoclax initiation provides effective tumor debulking, decreases the TLS risk category and reduces the need for hospitalization for intensive monitoring for TLS.

Supplementary Data from Effective Tumor Debulking with Ibrutinib Before Initiation of Venetoclax: Results from the CAPTIVATE Minimal Residual Disease and Fixed-Duration Cohorts

Purpose: Activating PIK3CA genomic alterations are frequent in head and neck squamous cell carcinoma (HNSCC), and there is an association between phosphoinositide 3-kinase (PI3K) signaling and radioresistance. Hence, we investigated the therapeutic efficacy of inhibiting PI3K with GDC-0032, a PI3K inhibitor with potent activity against p110α, in combination with radiation in HNSCC.Experimental Design: The efficacy of GDC-0032 was assessed in vitro in 26 HNSCC cell lines with crystal violet proliferation assays, and changes in PI3K signaling were measured by Western blot analysis. Cytotoxicity and radiosensitization were assessed with Annexin V staining via flow cytometry and clonogenic survival assays, respectively. DNA damage repair was assessed with immunofluorescence for γH2AX foci, and cell cycle analysis was performed with flow cytometry. In vivo efficacy of GDC-0032 and radiation was assessed in xenografts implanted into nude mice.Results: GDC-0032 inhibited potently PI3K signaling and displayed greater antiproliferative activity in HNSCC cell lines with PIK3CA mutations or amplification, whereas cell lines with PTEN alterations were relatively resistant to its effects. Pretreatment with GDC-0032 radiosensitized PIK3CA-mutant HNSCC cells, enhanced radiation-induced apoptosis, impaired DNA damage repair, and prolonged G2–M arrest following irradiation. Furthermore, combined GDC-0032 and radiation was more effective than either treatment alone in vivo in subcutaneous xenograft models.Conclusions: GDC-0032 has increased potency in HNSCC cell lines harboring PIK3CA-activating aberrations. Further, combined GDC-0032 and radiotherapy was more efficacious than either treatment alone in PIK3CA-altered HNSCC in vitro and in vivo. This strategy warrants further clinical investigation. Clin Cancer Res; 22(8); 2009–19. ©2015 AACR.

Supplementary Figure Legends

Supplementary Figure S1. GDC-0032 downregulated PI3K signaling in a dose dependent fashion. Supplementary Figure S2. BYL719 enhances radiation induced cell death in PIK3CA activated cell lines. Supplementary Figure S3. A66 enhances radiation induced cell death in PIK3CA activated cell lines Supplementary Figure S4. PI3K pathway inhibition enhances cell death in HSC-2 (PIK3CA mutated) following irradiation. Cells were treated with the given drugs for 24 hours, irradiated with 4 Gy, then assessed for Annexin V staining 72 hours after irradiation. Supplementary Figure S5. GDC-0032 radiosensitizes LB-771 (PIK3CA amplified) cells in vitro as assessed via the clonogenic survival assay. Supplementary Figure S6. GDC-0032 enhances DNA damage and apoptosis following irradiation. Supplementary Figure S7. GDC-0032 does not significantly alter the early G2/M checkpoint. Supplementary Figure S8. ATR inhibition partially blocks the early G2/M checkpoint. Supplementary Figure S9. Wee1 inhibition partially blocks the early G2/M checkpoint. Supplementary Figure S10. Wee1 inhibition doesn't reverse the enhanced G2/M checkpoint induced by GDC-0032 following irradiation. 3 Supplementary Figure S11. GDC-0032 and radiation is superior to either treatment alone in an HPV(+) oropharyngeal squamous cell carcinoma patient derived xenograft.

Head and neck squamous cell carcinomas have distinct mutation and copy-number profiles depending on human papillomavirus status. Although several challenges remain in biomarker implementation and clinical trial feasibility, incorporating available genomic data will expedite the development of novel therapeutics and predictive biomarker-driven clinical trials. Clin Cancer Res; 21(3); 495–7. ©2014 AACR. See related article by Seiwert et al., p. 632

Next generation sequencing technologies enable precise personalized medicine. Thus, patients with oncogene driven tumors are currently treated with targeted therapeutics such as EGFR inhibitors. However, drug interactions with other activated signaling pathways in treated tumors often alter predicted therapeutic response. Therefore, bioinformatics algorithms are needed to infer unanticipated molecular interactions from anticipated molecular response to targeted therapeutics in diverse genetic backgrounds. To model heterogeneous genetic backgrounds in HNSCC, we use HaCaT cells with forced overexpression of EGFR, HRAS, and PIK3CA. Previously, the CoGAPS matrix factorization algorithm was shown to infer the specific signaling pathways that were activated in these HaCaT knock-in constructs from gene expression data. In this study, we evaluated whether CoGAPS could also delineate unanticipated signaling changes from anticipated cellular signaling response caused by targeted therapeutic in diverse genetic backgrounds. To delineate these signaling responses, we measured gene expression after treating the modified HaCaT cells with three EGFR targeted agents (gefitinib, cetuximab and afatinib) for 24 hours. The CoGAPS matrix factorization algorithm distinguished a gene expression signature associated with the anticipated silencing of the EGFR network and a signature associated with unanticipated transcriptional feedback in HaCaT constructs that were sensitive to EGFR inhibitors. Notably, the feedback signature showed that EGFR gene expression itself increased in cells that were responsive to EGFR inhibitors. The CoGAPS algorithm further associated such feedback with increased expression of several growth factor receptors by the AP-2 family of transcription factors. Once transcribed, these growth factor receptors may ultimately compensate for EGFR inhibition in these sensitive cells. Our data suggest, that CoGAPS gene expression signatures delineate on target and feedback effects of drugs related to therapeutic sensitivity in diverse genetic backgrounds. Citation Format: Elana J. Fertig, Hiroyuki Ozawa, Manjusha Thakar, Jason Howard, Gabriel Krigsfeld, Alexander V. Favorov, Daria A. Gaykalova, Michael F. Ochs, Christine H. Chung. The CoGAPS matrix factorization algorithm infers feedback mechanisms from therapeutic inhibition of EGFR that increases expression of growth factor receptors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 777.

TNF-related apoptosis-inducing ligand (TRAIL; Apo2L) is an endogenous protein that induces tumor-selective apoptosis and is currently being tested in clinical trials as an anti-tumor agent. Despite promising preclinical activity, recombinant human TRAIL suffers from efficacy-limiting properties such as short serum half-life, stability, cost, and limitations in delivery, e.g. to the central nervous system. We hypothesized that induction of the human TRAIL gene by small molecules may overcome these limitations of TRAIL-based protein therapy. A cell-based luciferase reporter screen using the human TRAIL promoter identified TIC10 as a p53-independent small molecule inducer of the TRAIL gene. TIC10 induces TRAIL on the surface of normal and tumor cells, in sera of non-tumor-bearing mice, and throughout the tumor microenvironment. Co-culture experiments suggest that TRAIL upregulation by normal cells can contribute to the anti-tumor response of TIC10 through a TRAIL-mediated bystander effect. TIC10-induced TRAIL causes tumor-specific and TRAIL-mediated cell death as indicated by inhibition of cytotoxic effects following TRAIL gene knockdown in vitro and in vivo. Expression profiling of TIC10-induced transcriptional changes revealed altered expression levels of Foxo3a target genes, including DR5. We found that TIC10 causes the nuclear translocation of Foxo3a, but not Foxo1, to upregulate TRAIL gene transcription as determined by chromatin-immunoprecipitation assays. Western blot analysis indicated that TIC10 potently inhibits the phosphorylation of Foxo3a at Ser253 and Ser294 that normally docks the transcription factor to 14-3-3 proteins in the cytosol. Dual inhibition of Akt and ERK by TIC10, by other individual small molecule kinase inhibitors in combination or targeted siRNA inhibited the phosphorylation of these critical serine residues and synergistically induced Foxo3a-dependent TRAIL and TRAIL-mediated cell death. TIC10 is a promising first-in-class anti-tumor agent with a broad range of anti-tumor effects in preclinical animal models including othrotopic GBM, immunocompetent lymphoma, xenografted colon, lung, or breast cancers, and exhibits synergy with taxanes as well as bevacizumab among other agents. TIC10 appears to have no measurable toxic effects in mice including evaluation of serum chemistries, liver function studies, LDH, tissue histology, weight of the animals or behavioral changes. We are pursuing the clinical translation of these important findings that indicate a favorable and wide therapeutic index for a novel anti-tumor agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1935. doi:1538-7445.AM2012-1935

TRAIL is an attractive anti-neoplastic agent due to its minimal toxicity and its potent ability to induce apoptosis in cancer cells. Clinical trials with intravenous administration of recombinant human TRAIL in cancer patients have been limited by the short serum half-life of TRAIL and molecular resistance mechanisms including loss of cell surface death receptor expression and/or pro-survival expression changes in proteins that regulate apoptosis such as Mcl-1. Although TRAIL death receptor-targeted agonistic antibodies have an increased half-life, they act on one of the two pro-apoptotic TRAIL receptors DR4 (TRAIL-R1) or DR5 (TRAIL-R2). In order to pharmacologically increase tumor and host TRAIL production as a novel anti-neoplastic mechanism, to improve the pharmacokinetic properties of TRAIL by increasing its effective half-life, and perhaps its local concentration within the tumor microenvironment, we screened for small molecules capable of inducing the endogenous TRAIL gene. A cell-based bioluminescence reporter screen was conducted in TRAIL-resistant Bax-null human HCT116 colon cancer cells using the TRAIL gene promoter yielded TRAIL-inducing compound 10 (TIC10). TIC10 induces TRAIL at the message, membrane-bound, and soluble levels in a time- and dose-dependent manner in TRAIL-sensitive and TRAIL-resistant human cancer cell lines as well as normal human fibroblasts without any effect on cell cycle profile. TIC10 induces cell death in a number of cancer cell lines without evidence of cell cycle arrest, a characteristic of HDAC inhibitors that are the only other pharmacological agent currently shown to upregulate TRAIL. Lack of structural homology with HDAC inhibitors along with this observation suggest a novel mechanism of TRAIL-upregulation. Western blot analysis revealed that TIC10 sensitizes to TRAIL-mediated apoptosis by tumor cell-specific upregulation of DR5 and downregulation of Mcl-1. Human xenograft experiments demonstrate that TIC10 is highly effective as a multi- or single-dose agent with similar efficacy to TRAIL in TRAIL-sensitive xenografts. Some TRAIL-resistant xenografts such as RKO demonstrate TIC10 superiority to TRAIL with no incidence of toxicity. TIC10 causes highly increased protein levels of TRAIL and cleaved caspase-8, increased TUNEL staining indicating apoptosis, in addition to striking decrease in tumor cell density by histology in tumors of human xenografts in mice. Mice lacking tumors demonstrate an upregulation of TRAIL protein levels in serum. The effect of TIC10 on normal cell production of TRAIL provides a novel mechanism for killing tumor cells through a bystander effect in the tumor microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4502. doi:10.1158/1538-7445.AM2011-4502

TNF-related apoptosis-inducing ligand (TRAIL) has been hailed as a promising cancer drug target due to its apoptosis-inducing capabilities which are aimed selectively at cancer cells. Methods of inducing endogenous TRAIL as well as introducing exogenous recombinant human TRAIL (rhTRAIL) are being explored in preclinical studies and in the case of the latter, clinical trials. While the gene encoding for DR5, a TRAIL death receptor, has been known to be regulated by p53, we have recently shown that TRAIL is regulated by p53 at base pairs 504-1102 of its promoter (Kuribayashi et al., Cancer Biology and Therapy. 7:2034-2038, 2008). p53 is mutated in over half of all cancers; therefore, a method of upregulating endogenous TRAIL via a mechanism independent of p53 holds great potential therapeutic value for cancer. We searched for small molecule p53-independent upregulators of TRAIL using a luciferase reporter assay which utilized a plasmid with the gene encoding for firefly luciferase under the transcriptional control of the first 504 base pairs of the human TRAIL promoter. Screening with the NCI Diversity Set II library yielded several hits with >2-fold TRAIL induction which were repeated and further analyzed for use as a drug and to gain mechanistic insight. Interestingly, one hit from this screen was breflate, the prodrug of Brefeldin A (BFA). BFA inhibits retrograde protein transport from the Golgi apparatus to the endoplasmic reticulum and is currently being investigated as a cancer therapeutic. Shao et al. (Exp. Cell Res. 227:190-196, 1996) have previously reported that BFA is a potent inducer of apoptosis in some cancer cell lines independently of p53, though the mechanistic relationship was not clear. Breflate clearly increased intracellular levels of TRAIL in HCT116 Bax−/− cells as determined by Western blot analysis, and in addition to the original reporter assay this is being corroborated by reverse transcriptase polymerase chain reaction (RT-PCR). Moreover, sub-G1 analysis found that breflate induced levels of apoptosis >2-fold relative to control levels at 10μM after 24 hours in HCT116 p53−/− cells. Further mechanistic and ongoing in vivo studies are being conducted with breflate alone and in combination with chemotherapy or targeted cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5467.