Clinical Activity In Phase Research Articles

Cyclin-Dependent Kinase Inhibitor P1446A Induces Apoptosis in a JNK/p38 MAPK-Dependent Manner in Chronic Lymphocytic Leukemia B-Cells

Regimented activation of cyclin/CDK complexes is required for orderly cell cycle progression. Targeting this process has been an attractive therapeutic approach in neoplasia due to cell cycle deregulation and excessive proliferation. CDK inhibitors (notably flavopiridol and dinaciclib) have shown remarkable efficacy in CLL, attributed to their effect on CDK7/9 which is accompanied by a disruption of RNA polymerase-mediated transcription and subsequent deregulation of short-lived pro-survival proteins such as MCL1. Recently, endoplasmic reticulum (ER) stress has been implicated in CDK inhibition in CLL. A novel orally active kinase inhibitor P1446A (Piramal Healthcare Ltd) inhibits CDKs at nM concentrations and has demonstrated clinical activity in Phase I studies in solid tumors with minimal hematologic toxicity. Here we conducted a pre-clinical study of P1446A in CLL B-cells.Peripheral blood cells were obtained from patients with CLL and isolated using standard techniques. 83.3±3.3% of CLL cells underwent apoptosis upon 24-hour exposure to 1.5 µM P1446A, irrespective of chromosomal abnormalities [del(17p)/11q] or IGHV mutationalstatus. Apoptosis occurred as early as 4 hours after drug treatment and was accompanied by a dose-dependent inhibition of interphase CDKs (2/4/6) as evidenced by reduced phospho-Rb (T821/ S780). Apoptosis preceded reduction in RNA polymerase phosphorylation and thus occurred before transcriptional CDK7/9 were inhibited. Meanwhile, targeting CDK2 and CDK4/6 using specific inhibitors did not induce cell death in CLL.We supposed that P1446A could lead to ER stress induction in primary CLL cells, culminating in apoptosis. We noted rapid activation of JNK and p38 MAPK in CLL cells treated with P1446A in vitro. Phosphorylation of JNK and p38 MAPK occurred within 2 hours of treatment with P1446A, preceded onset of apoptosis as measured by PARP cleavage, and was followed by induction of activating transcription factors 2 and 3 (downstream targets of JNK/p38MAPK). Pharmacologic inhibitors of JNK/p38 conferred partial protection from P1446A-mediated apoptosis. We sought to identify the upstream regulators of JNK in this setting. Rapid phosphorylation of ASK1 at Thr845, a site required for its kinase activity, occurred in CLL cells treated with P1446A in a time and dose-dependent manner, concurrently with JNK activation. We observed formation of the trimeric complex between ASK1, IRE1, and TRAF2. Genetic knockdown of ASK1 led to a reduction in JNK phosphorylation and a partial rescue from apoptosis. P1446A did not induce unfolded protein response (UPR) beyond ASK1/JNK/p38 activation, consistent with previous reports on its limited functionality in CLL cells. By contrast, we found that treatment with P1446A led to a dramatic induction of NOXA mRNA and protein levels, in a JNK-dependent manner.In summary, we demonstrate that CDK inhibitor P1446A is a potent inducer of apoptosis in primary CLL cells in vitro. P1446A leads to partial activation of ER stress and the UPR in CLL cells manifested by ASK1-dependent signaling, leading to JNK/p38 MAPK activation and up-regulation of NOXA. This study provides rationale for additional investigations of P1446A in CLL. DisclosuresSpurgeon:Bristol Meyers Squibb: Research Funding; Acerta Pharma: Research Funding; Gilead sciences: Honoraria, Research Funding; Genentech: Honoraria; Pharmacyclics: Honoraria; Janssen: Research Funding. Srinivasa:Piramal Healthcare: Employment.

Phase II studies of nivolumab (anti-PD-1, BMS-936558, ONO-4538) in patients with advanced squamous (sq) or nonsquamous (non-sq) non-small cell lung cancer (NSCLC).

8027 Background: Nivolumab, a fully human IgG4, PD-1 immune-checkpoint inhibitor antibody, has shown durable clinical activity in phase I and II trials in several tumor types. Recently, a phase III...

Recently Developed Therapeutic Agents for B-cell Non-Hodgkin Lymphoma

While most B-cell lymphomas are cured by chemotherapy, in recent years research interest has focused on the development of monoclonal antibodies and small molecules targeting membrane proteins, components of signaling pathways or tumor microenvironment, which are factors essential for the development and progression of lymphomas. Some of these new drugs have shown substantial clinical activity in phase I and small phase II studies in patients with relapsed and refractory disease, with response rates of up to 50-60% in some cases. These results give hope for patients who have standard treatment failure; however, their incorporation into commonly used regimens also represents a significant challenge for the future. (Korean J Med 2015;88:247-251)

Extracellular signal-regulated kinase 5 and cyclic AMP response element binding protein are novel pathways inhibited by vandetanib (ZD6474) and doxorubicin in mesotheliomas.

Malignant mesothelioma (MM), lung cancers, and asbestosis are hyperproliferative diseases associated with exposures to asbestos. All have a poor prognosis; thus, the need to develop novel and effective therapies is urgent. Vandetanib (Van) (ZD6474, ZACTIMA) is a tyrosine kinase inhibitor that has shown equivocal results in clinical trials for advanced non-small cell lung cancer. However, tyrosine kinase inhibitors alone have shown no significant clinical activity in phase II trials of patients with unresectable MM. Using epithelioid (HMESO) and sarcomatoid (H2373) human MM lines, the efficacy of tumor cell killing and signaling pathways modulated by Van with and without doxorubicin (Dox) was examined. Van alone reduced total cell numbers in HMESO MM and synergistically increased the toxicity of Dox in HMESO and H2373 cells. Most importantly, we identified two novel cell survival/resistance pathways, ERK5 and cyclic AMP response element binding protein (CREB), that were inhibited by Van and Dox. After silencing of either ERK5 or CREB, significant decreases in cell numbers in the Dox-resistant sarcomatoid H2373 line were observed. Results suggest that a plethora of cell signaling pathways associated with cell survival are induced by Dox but inhibited by the addition of Van in MM. Data from our study support the combined efficacy of Van and Dox as a novel approach in the treatment of MM that is further enhanced by blocking ERK5 or CREB signaling cascades.

83IN - Evolving Biology of Sarcoma

ABSTRACT The biology of soft tissue sarcomas and locally aggressive connective tissue tumors evolves rapidly. At least seven molecular subgroups of connective tissue tumors may be distinguished: 1) sarcoma with specific translocations generating fusion transcription factors or growth factors; 2) sarcomas with mutated activated kinases (KIT in GIST); 3) sarcomas with deletion of tumor suppressor genes such as NF1 sarcomas; 4) Sarcomas with simple genetic alterations (MDM2/CDK4 gene amplification in WD/DD liposarcomas;); 5) Tumors with alterations of the intercellular adhesion pathways (aggressive fibromatosis with APC deletion or beta catenin mutations); 6) tumors with histone protein mutations such as giant cell tumor of the bone; 5) Finally, sarcomas with gross genetic alterations (e.g. leiomyosarcomas, UPS) represent a mixed bag for which the CINSARC expression profile distinguishes subgroups with very different prognosis This biological classification is therefore rapidly evolving and guides in real time the development of novel treatment. The identification of the “driver” mutation paved the way for the selection of efficient targeted therapies, following the paradigm of KIT and PDGFRA kinase mutations in GIST. Other examples are currently identified, in DFSP, a PDGF driven tumor and in PVNS, an M-CSF driven tumor. For this later group, 2 novel MCSFR inhibitors have been shown to have an outstanding clinical activity in phase I in ASC O 2014. This concept has therefore been fruitful, but now shows limitations, as observed with IGF1R Ab, or may be associated with a less dramatic activity in some pathways such as MDM2 inhibitors or CDK4 inhibitors. In addition, tumor heterogeneity, in the primary tumor, in the metastases, and following exposure to a TKI are now frequently observed, calling for innovative strategies to overcome this common phenomenon. During this presentation, we will review how the understanding of the driver molecular alterations of these tumors paved the way for the development of targeted treatment inhibiting driver mutations, and discuss the next steps to be explored. Disclosure: JY Blay: Research support and honoraria from Novartis, Roche, Bayer, Pharmamar and GSK.

Preliminary results of a phase II randomized study (ROMULUS) of polatuzumab vedotin (PoV) or pinatuzumab vedotin (PiV) plus rituximab (RTX) in patients (Pts) with relapsed/refractory (R/R) non-Hodgkin lymphoma (NHL).

8519 Background: PoV and PiV, antibody drug conjugates (ADC) containing the anti-mitotic MMAE targeting CD79b (PoV) and CD22 (PiV), showed clinical activity in Phase I. The current study aims to compare PoV and PiV + RTX in R/R DLBCL and R/R follicular lymphoma (FL). Methods: Pts were randomized to receive PoV or PiV + RTX (ADC 2.4 mg/kg + RTX 375 mg/m2) every 21 days. Tumor assessments were performed every 3 months. Results: As of 8 November 2013, 58 received PoV + RTX (38 DLBCL; 20 FL), 63 PiV + RTX (42 DLBCL; 21 FL). Median prior therapies [DLBCL, 3 (1-10); FL, 2 (1-8)] were balanced between treatment arms; 46% were RTX refractory. Median treatment (tx) cycles in DLBCL: 5 for both ADC (1-15); FL: 8.5 PoV (3-15) and 6 PiV (1-13). Overall safety profiles of both regimens were similar. Tx-emergent adverse events (AE) >25%: fatigue (52%), diarrhea (42%), nausea (37%), peripheral neuropathy (PN) (32%), constipation (26%). Grade ≥ 3 AE >3%: neutropenia (21%), diarrhea (6%), dyspnea (4%), febrile neutropenia ...

Immunoglobulin-like transcript 2 (ILT2) is a biomarker of therapeutic response to oncolytic immunotherapy with vaccinia viruses.

BackgroundOncolytic viruses represent a novel form of cancer immunotherapy. Vaccinia viruses encoding human T cell co-stimulatory molecules have demonstrated clinical activity in phase I clinical trials in patients with advanced melanoma. However, predictive biomarkers of therapeutic response have not yet been identified.MethodsA customized microarray was performed to identify changes in peripheral blood mononuclear cell (PBMC) gene expression upon exposure to recombinant oncolytic vaccinia viruses. Up-regulated and down-regulated genes were identified and selected for further analysis using PBMC samples from normal donors and oncolytic virus-treated patients before and after viral injection. Quantitative PCR and flow cytometry of defined T cell subsets was performed to evaluate expression patterns and clinical correlations.ResultsThe microarray identified 301 genes that were up-regulated and 960 genes that were down-regulated in T cells after exposure to oncolytic vaccinia virus. The B7.1 gene was highly up-regulated and the immunoglobulin-like transcript 2 (ILT2) gene was highly down-regulated by vaccinia-B7.1, which was consistent with the known inverse regulation of these two genes. We observed an inverse association between ILT2 expression in the tumor microenvironment and clinical response and further identified ILT2 as a marker of regulatory CD4+ and suppressor CD8+ T cell responses and whose down-regulation was predictive of therapeutic responses in patients treated with oncolytic virus immunotherapy.ConclusionsILT2 is a new putative biomarker of T cell and clinical response in patients treated with oncolytic vaccinia virus immunotherapy. Further confirmation of ILT2 as a biomarker requires prospective validation in a larger series of clinical trials.

Chronic Myeloid Leukemia— What Else is there Beyond Protein Kinase Inhibitors?

Chronic myeloid leukemia (CML) is induced by the BCR-ABL oncogene. The advent of BCR-ABL tyrosine kinase inhibitors (TKIs) has redefined treatment goals in CML starting with imatinib and followed by the newer TKI inhibitors including dasatinib, nilotinib, bosutinib, and ponatinib. However, a significant proportion of patients do not achieve a satisfactory response to TKIs and resistance remains an unmet need in the in the treatment of CML. Furthermore, disease eradication with TKIs may pose a significant challenge: minimal residual disease (MRD) remains detectable following treatment with these agents. Recently, several new molecular targets have been proposed in CML, and several drugs are in clinical development. Omacetaxine, a protein translation inhibitor, has shown the potential to substantially reduce MRD in animal models and has demonstrated clinical activity in phase II clinical trials regardless of patients’BCR-ABLT313I mutation status. The broad range of therapeutic effects associated with interferon may reduce resistance and relapse, and has resulted in a resurgence of interest in this therapy. In addition, several other therapeutic targets are currently undergoing clinical investigation.

Ibrutinib (PCI-32765) Antagonizes Rituximab-Dependent NK-Cell Mediated Cytotoxicity

IntroductionIbrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (BTK) with promising clinical activity in phase I/II clinical trials in CD20+ B-cell malignancies for which rituximab-combination chemotherapy is today's standard of care. Given homology between BTK and interleukin-2 inducible tyrosine kinase (ITK), we recently confirmed based on molecular and phenotypic analysis that ibrutinib irreversibly binds ITK (Dubovsky J, et al: Blood 2013). ITK expression in FcR-stimulated NK cells leads to increased calcium mobilization, granule release, and cytotoxicity (Khurana D et al: J Immunol 2007). As FcR stimulation is requisite for antibody-dependent NK-cell mediated cytotoxicity (ADCC), we investigated if ibrutinib influenced rituximab's anti-lymphoma activity as well as trastuzumab's anti-HER2+ breast cancer activity. MethodsRituximab and trastuzumab-dependent NK-cell mediated cytotoxicity was assessed using lymphoma and HER2+ breast cancer cell lines. In vitro NK cell cytokine secretion, degranulation and cytotoxicity were assessed by IFN-g release, CD107a mobilization and chromium release. Xenotransplant lymphoma and breast cancer models in athymic, nu/nu mice were used to assess efficacy of ibrutinib and rituximab or trastuzumab combination regimens. Using an autologous system from patients with chronic lymphocytic leukemia (CLL), inhibition of NK cell function was compared between ibrutinib and CGI-1746 (Di Paolo JA, et al: Nature Chem Biol 2011), a previously reported selective BTK inhibitor lacking ITK inhibitory function. ResultsFcR-stimulated NK cells following exposure to rituximab-coated lymphoma cells express high and moderate levels of ITK and BTK, respectively. Ibrutinib inhibited both rituximab- and trastuzumab-induced NK cell cytokine secretion in a dose-dependent manner at 0.1 and 1mM of ibrutinib in vitro (Fig 1A *p=.009, **p=.001, ***p<.001). Similarly, ibrutinib prevented FcR-stimulated NK cell degranulation by ∼60% and ∼90% at 0.1 and 1mM (Fig 1B *p=.013, **p=.017, ***p=.002, ****p=.001). In vitro cytotoxicity of rituximab-coated, chromium-labeled lymphoma cells was observed in an NK cell-independent and ibrutinib-concentration dependent manner (Fig 1C *p<.001, **p=.045). In contrast, in vitro cytotoxicity of trastuzumab-coated, chromium-labeled breast cancer cells was inhibited in an ibrutinib dose-dependent manner (Fig 1C ***p=.036, ****p=.010). Abrogation of trastuzumab-dependent NK cell-mediated cytotoxicity was confirmed in vivo with concurrent ibrutinib daily dosing for 2 weeks during trastuzumab treatment, 4 doses, as measured by tumor growth and survival (Fig 2A, *p<.001; Fig 2B *p=.18). Concurrent ibrutinib daily dosing for 2 weeks during 4 doses of rituximab therapy, similarly, antagonized rituximab's efficacy in tumor growth and survival, with anti-lymphoma activity of the combination equivalent to ibrutinib monotherapy (Fig 2C *p=.049, **p=.032; Fig 2D *p=.29). Sequential ibrutinib for 1 week followed by 2 doses of rituximab, or sequential rituximab (2 doses) followed by ibrutinib for 1 week resulted in restored anti-lymphoma activity superior to concurrent combination therapy of ibrutinib for 2 weeks and 4 doses of rituximab (Fig 2E *p<.001; Fig 2F *p<.001). Autologous CLL samples, n=6, confirmed similar effect as observed with lymphoma cell lines of 0.1 and 1mM of ibrutinib resulting in in vitro inhibition of NK cell degranulation and cytotoxicity by ∼65% and ∼85%, respectively. Interestingly, in vitro, CGI-1746 had no antagonistic effect on ADCC against rituximab treated targets (CLL cells and Raji lymphoma cell line). [Display omitted] [Display omitted] ConclusionsPreclinical investigation of a combination therapy currently in Phase II and III clinical trials, adding a promising novel agent, ibrutinib, to a current standard of care, rituximab, for the treatment of B-cell lymphomas, surprisingly results in antagonistic effects. We demonstrate that the abrogation of both rituximab's and trastuzumab's anti-tumor efficacy is a result of ibrutinib's inhibition of FcR-stimulated NK cell function, specifically ADCC. Selective BTK inhibitors or alternative ibrutinib dosing schedules, sequential versus concurrent, may preserve the anti-lymphoma efficacy of both agents. Disclosures:Buggy:Pharmacyclics: Employment, Equity Ownership.

A Novel Cyclin Dependent Kinase Inhibitor P1446A Induces Apoptosis Of Chronic Lymphocytic Leukemia B Cells

Cyclins are important regulators of cell cycle in neoplastic cells. Regimented fluctuation in their expression is required for orderly cell cycle progression. Cyclins partner and modulate cyclin-dependent kinases (CDK) which are necessary for their activity. Since neoplastic cells are characterized by deregulated cell cycle with excessive proliferation, targeting cell cycle progression has been an attractive therapeutic approach. CDK inhibitors, most notably flavopiridol and dinaciclib, have shown efficacy in early phase clinical trials in CLL. The dominant fraction of the CLL clone is represented by B-cells in a resting (G0) state of the cell cycle. Efficacy of the CDK inhibitors in CLL is attributed to their effect on CDK-7/9 which facilitate initiation and sustenance of RNA polymerase-mediated transcription. P1446A is a novel potent and orally active kinase inhibitor which inhibits CDK's at nanomolar concentrations (CDK-1 IC50 25 nM, CDK-4 IC50 90 nM, CDK-6 IC50 210 nM and CDK-9 IC5022 nM). P1446A has shown clinical activity in Phase I studies in solid tumors and exhibited minimal hematologic toxicity. Here we studied activity of P1446 in primary CLL B-cells and MEC1 cells.We enrolled 40 patients with median age of 70 years at the CLL clinic at Dartmouth. Median time of follow-up was 4 years and most patient (75%) were untreated. 60% of patients had unmutated IGHV. The majority of patients had normal cytogenetics, trisomy 12 or 13q deletion, and 3 patients (10%) had deletion 11q. Additionally, 10 patients with deletion 17p were enrolled at the CLL Center at Dana Farber Cancer Institute. CLL B cells were isolated from peripheral blood using standard Ficoll-Hypaque technique and cultured in RPMI supplemented with 15% fetal bovine serum. Apoptosis was quantified by means of Annexin V/7-AAD staining and flow cytometry as well as immunoblotting (PARP cleavage). B-cell receptor simulation was performed with 10 mg/mL immobilized anti-IgM. We also determined activity of P1446 in MEC1 cells.CLL cells showed features of apoptosis (PARP cleavage) after a 2-hour incubation with 1500 nM P1446A. While apoptosis began to occur with lower concentrations of P1446A (0.2 μM), maximal efficacy was reached with 1.5 μM. After 24 hours of incubation with that dose, 84.1±4.7% CLL cells demonstrated Annexin V staining, compared to 30.2±2.5% of cells treated with vehicle control (p<0.001). P1446A-induced cell death did not depend on the IGHV mutational status (p=0.44) or cytogenetic abnormalities. However, samples which carried deletion 17p showed decreased sensitivity towards the CDK inhibitor compared with those which had normal cytogenetics or non-17p abnormality (p=0.003). Still, 37.5±4.4% cells carrying 17p del underwent apoptosis upon 24-hour exposure to 1.5 μM P1446 (normalized to untreated control). P1446A was significantly more toxic to CLL cells compared to normal B-cells (p<0.001). To determine whether P1446A had an impact on BCR signaling-mediated survival, we co-cultured CLL cells with 10 mg/mL immobilized anti-IgM. As expected, BCR engagement resulted in CLL cell rescue from spontaneous apoptosis in a subset of patient samples, while co-incubation with P1446A abrogated this protective effect.Similarly, P1446 induced significant apoptosis in MEC1 cell line leading to apoptosis induction after 6 hours of incubation with 1.5 μM P1446A. We determined that 48-hour incubation with 3 μM P1446A resulted in a two-fold reduction of MEC1 metabolic activity in an MTT assay.Our data shows that a novel CDK inhibitor P1446A demonstrates significant pre-clinical activity in CLL. Disclosures:Agarwal:Piramal Enterprizes Ltd.: Employment.

An open-label, randomized, phase II study of nivolumab (anti-PD-1; BMS-936558; ONO-4538) given sequentially with ipilimumab in patients (pts) with advanced or metastatic melanoma (MEL).

TPS9107^ Background: Programmed death-1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) are immune checkpoint receptors that attenuate T-cell responses and contribute to immune evasion by tumor cells. Nivolumab is a PD-1 receptor blocking monoclonal antibody that has shown clinical activity in phase 1 trials with various tumor types including MEL. Ipilimumab is an anti-CTLA-4 monoclonal antibody approved for advanced or metastatic MEL. Preclinical data suggest that combined PD-1 and CTLA-4 receptor blockade may improve antitumor activity in MEL, with PD-1 blockade upregulating CTLA-4 expression and CTLA-4 blockade upregulating PD-1 expression on tumor-infiltrating T cells. We describe a phase II study evaluating nivolumab administered sequentially with ipilimumab in pts with advanced (unresectable stage III) or metastatic (stage IV) MEL. Methods: This open-label, phase II study will enroll approximately 80 pts. During the induction period, pts will be randomized 1:1 to either nivolumab (3 mg/kg, q2w) from Weeks (Wk) 1-13 followed by ipilimumab (3 mg/kg, q3w) from Wks 14-25, or ipilimumab followed by nivolumab at the same doses and schedules. All pts will then receive nivolumab (3 mg/kg, q2wk) during the continuation period until disease progression or unacceptable toxicity, for ≤2 years from initial study treatment. Pts may be treatment-naive or have disease recurrence or progression after one prior systemic therapy, excluding prior anti-PD-1, anti-CTLA-4, or a BRAF inhibitor. The primary endpoint is the incidence of treatment-related Grade 3–5 adverse events (AEs) during the induction period. Secondary endpoints are response rate at Wk 25 and progression rates at Wks 13 and 25. Exploratory endpoints include safety and tolerability; overall survival; pt-level time course of treatment-related grade 3–5 AEs, response, progression, treatment discontinuation, and death; the association of changes in pharmacodynamic immune markers from baseline to Wk 13 and Wk 25 and clinical response; the association between baseline immunological parameters and clinical response; and immune-related single nucleotide polymorphisms. Clinical trial information: NCT01783938.

A phase I study of lirilumab (BMS-986015), an anti-KIR monoclonal antibody, administered in combination with ipilimumab, an anti-CTLA4 monoclonal antibody, in patients (Pts) with select advanced solid tumors.

TPS3106 Background: Immune checkpoint blockade represents a novel form of cancer immunotherapy. Killer cell immunoglobulin-like receptors (KIR) and cytotoxic T lymphocyte antigen-4 (CTLA-4) are immune receptors that down-regulate NK and T cell activity, respectively. The anti-KIR antibody, lirilumab (BMSE986015), potentiates innate immunity by blocking signaling through inhibitory KIRs and has demonstrated modest side effects in a Phase I trial. The anti-CTLA-4 antibody, ipilimumab, potentiates adaptive immunity and has demonstrated improved overall survival in pts with advanced melanoma and preliminary evidence of clinical activity in Phase I and II trials. We hypothesized that coordinate modulation of innate and adaptive immunity by combining anti-KIR and anti-CTLA4 antibodies could achieve enhanced biologic and clinical activity compared to either agent alone. Here, we describe a Phase I study of lirilumab plus ipilimumab in pts with selected advanced solid tumors. Methods: This study will be performed in two parts and enroll approximately 150 pts. During dose escalation, pts with advanced melanoma, non-small cell lung cancer and castrate resistant prostate cancer, will be enrolled. During cohort expansion, 20 pts with each tumor type will be enrolled at the maximum tolerated dose (MTD), or the maximum administered dose, if no MTD is defined. The primary study objectives are to delineate the safety and tolerability, dose limiting toxicities, and MTD of this combination. Secondary objectives are to assess preliminary anti-tumor activity, pharmacokinetics, and immunogenicity of this combination in all pts, and the pharmacodynamic effects on tumor infiltrating lymphocytes in a cohort of melanoma pts. Exploratory objectives include a thorough assessment of the modulation of innate and adaptive immunity by this combination in peripheral blood and/or tumor specimens, and preliminary evaluation of the association of these changes with clinical outcome. Clinical trial registration number: NCT01750580 Clinical trial information: NCT01750580.

Ibrutinib Is an Irreversible Molecular Inhibitor of Interleukin-2 Inducible Kinase: Expanding Therapeutic Potential and Modulating a Th1 Selective Pressure in CD4 T-Cells

AbstractAbstract ▪775▪This icon denotes a clinically relevant abstractIn chronic lymphocytic leukemia (CLL), mounting evidence points to an aberrant tumor associated Th2 bias that drives leukemic cell immune evasion, promotes formation of a supportive niche microenvironment, and functionally cripples innate and adaptive immunity. The end result is a high incidence of infections which is the primary cause of mortality in CLL. This same Th2 bias is induced by many other types of cancer. Th2 CD4 T-cells are singularly dependent upon IL-2-inducible T-cell kinase (ITK) for activation whereas Th1 CD4 and CD8 T-cells have compensatory resting lymphocyte kinase (RLK) which conducts T-cell receptor activation even in the absence of ITK. Thus, a clinically viable ITK inhibitor would be ideal for targeting immune suppression associated with CLL and potentially other types of cancer. Unfortunately, no such therapeutic is currently available. Ibrutinib, a confirmed inhibitor of the Bruton’s tyrosine kinase (BTK) that irreversibly blocks downstream B-cell receptor activation, has demonstrated outstanding clinical activity in phase I/II clinical trials resulting in durable remissions in CLL. Our studies unveiled a previously uncharacterized Th1 cytokine switch in ibrutinib treated CLL patients which could not be attributed to B-lymphocytes. This ibrutinib-induced Th1 T-cell skewing was confirmed using the EμTCL1 mouse model of leukemia. Such alterations in cytokine patterns were reminiscent of mouse studies in which genetic ablation of ITK subverted Th2 immunity, thereby potentiating Th1-based adaptive immunity. The striking homology between BTK and ITK combined with intriguing in silico docking studies and promising in vitro kinase inhibition profiles with ibrutinib led to the hypothesis that this could be the first clinically viable irreversible ITK inhibitor. Cellular probe assays confirmed that the active site of ITK was covalently blocked by ibrutinib at pharmacologically relevant doses. Our comprehensive molecular analyses of T-cell signaling confirmed this in the Jurkat cell line. We further confirmed both molecular and functional outcomes in primary and in vitro polarized Th1 and Th2 CD4 T-cells. We found that mutation of the ITK-Cys442 covalent binding residue for ibrutinib alleviated molecular inhibition. We also demonstrated that Th1 and CD8 T-cell restricted expression of RLK provides a compensatory platform for T-cell activation offering a molecular explanation for the selective outgrowth of cytotoxic Th1 biased immunity. We further confirmed this effect using T-cells directly derived from CLL patients. To demonstrate that ibrutinib-induced ITK inhibition had direct clinical relevance in the setting of CLL we utilized a novel listeriosis/leukemia mouse model. In this model we clearly demonstrated complete recovery of functional immunity and all ibrutinib treated mice survived a potentially lethal Listeria monocytogenes infection. Our results expose novel molecular insights into the mechanism of action of ibrutinib in the context of Th2-biased immunosuppressive leukemia. We also postulate that ibrutinib’s irreversible ITK inhibitory effects may prove effective in a number of other autoimmune, inflammatory, and viral diseases, including influenza A and HIV/AIDS. Disclosures:Jaglowski:Pharmacyclics: Research Funding. Chang:Pharmacyclics, Inc.: Employment. Maddocks:Pharmacyclics: Research Funding. Buggy:Pharmacyclics: Employment, Equity Ownership. Byrd:Pharmacyclics: Research Funding.

Bruton's Tyrosine Kinase (BTK) Inhibitor Ibrutinib (PCI-32765) Blocks Hairy Cell Leukemia (HCL) Survival, Proliferation, and BCR Signaling: A New Therapeutic Approach for HCL

AbstractAbstract 1802Hairy cell leukemia (HCL) is an indolent B-cell malignancy characterized by marked splenomegaly, progressive pancytopenia, and BRAF V600E mutation as a disease-defining genetic event. Currently, treatment with purine nucleoside analogues lead to remission in nearly all patients, however most patients eventually progress and may became purine analogue-resistant after multiple courses; therefore, new targeted therapy approaches for HCL are needed. B cell receptor (BCR) signaling is involved in pathogenesis and progression of several B-cell malignancies. Upon antigen stimulation, the BCR initiates activation of a signaling cascade including the kinases Lyn and Syk, causing downstream BTK activation. BTK is also known to be involved in signaling of chemokine receptors (CXCR4, CXCR5) and adhesion molecules that are important for B-cell migration and tissue homing. Ibrutinib (PCI-32765) is a selective, irreversible BTK inhibitor (IC50= 0.5 nM) that has demonstrated clinical activity in phase I and II studies in patients with non-Hodgkin lymphoma, particularly in patients with chronic lymphocytic leukemia and mantle cell lymphoma.The importance of BTK in BCR signaling in hairy cell leukemia has not yet been characterized. Here, we explored the role of BTK and the activity of Ibrutinib in BCR signaling in HCL cell lines and primary HCL cells. We found BTK expression at the protein level in HCL cell lines (ESKOL and HC-1) and RAMOS control B cells. In cell growth and XTT assays, we found a significantly inhibition of HCL growth and proliferation with Ibrutinib at 0.5 μM, 1 μM and 5 μM concentrations in a dose-dependent manner. In accordance with these findings, we noticed a dose dependent S phase reduction by Ibrutinib, assessed by BrdU incorporation. Ibrutinib had no effects on HCL cell line viability in apoptosis assays after 24 or 48hs of incubation with Ibrutinib. Next, we tested effects of Ibrutinib on signaling after BCR triggering with anti-IgM or anti-IgA. Treatment with 1μM Ibrutinib reduced levels of BCR-induced phospho-ERK and phospho-AKT. Also, we demonstrated significant reductions in levels of BCR-induced chemokines (CCL3, CCL4) in HCL supernatants, which function as a surrogate marker for BCR activation. Primary HCL cells from 8 patients were tested for Ibrutinib-induced apoptosis. Here, we found a significantly reduction in HCL viability after 48 h of incubation, as shown in the figure. Similar results were obtained with the XTT assay, which was performed in parallel. To study effects of Ibrutinib on BCR signaling and activation in primary HCL cells, we performed immunoblot analysis after BCR triggering and measured CCL3 and CCL4 levels in HCL supernatants. Ibrutinib inhibited activation of BTK, ERK and ATK and significantly reduced levels of CCL3 and CCL4 (see Figure). Collectively, our studies demonstrate that Ibrutinib inhibits BCR signaling in primary HCL cell and HCL cell lines, and significantly diminishes HCL survival and proliferation. These data provide a strong rationale for testing the clinical activity of this new, targeted and well-tolerated agent in patients with HCL. [Display omitted] Disclosures:Buggy:Pharmacyclics: Employment, Equity Ownership. Burger:Pharmacyclics: Consultancy, Research Funding.

78IN - Is there a Role for Targeted Agents or Biologicals in Stage I–III NSCLC?

ABSTRACT More than half of the patients with non-small cell lung cancer (NSCLC), including different tumor stages and histology subtypes, have actionable driver mutations. In patients with advanced NSCLC and activating EGFR mutations, randomized phase III trials with EGFR inhibitors (gefitinib, erlotinib and afatinib) demonstrated superior response rates and progression free survival compared with chemotherapy. Phase II trials showed high response rates for crizotinib in advanced NSCLC with ALK-translocation and the results of two randomized phase III trials are awaited. Preliminary data from phase I-II trials and retrospective series suggest further targets for tumor-specific therapies, including MET, ROS1, HER2, BRAF, KRAS, PI3K and others. Moreover, bevacizumab and cetuximab have shown clinical activity in phase III trials in combination with chemotherapy in patients with advanced NSCLC selected by histology and EGFR expression, respectively. Rapid molecular testing is now available at many institutions, and the translation of these novel therapies from stage IV to earlier stages has begun. Caution is warranted, because cure – not tumor response – is the ultimate goal of therapy, and the use of new drugs outside of a clinical trial in patients with stage I-III NSCLC may be risky. Of note, S0023 and BR.19 showed no benefit with the addition of gefitinib to a radical treatment in patients with localized NSCLC, irrespective of EGFR mutation status. Furthermore, phase III trials suggested a possible antagonism when EGFR-TKIs were combined with chemotherapy in patients with stage IV NSCLC. More recent studies indicate that such negative interaction may be avoided by pharmacodynamic separation (or intercalation), and that EGFR-TKIs as single agents could be used safely and effectively as induction therapy before surgery in carefully selected patients with stage I-III NSCLC. At the meeting, these data will be reviewed, and ongoing trials in this field will be discussed, including RADIANT (erlotinib), ADJUVANT (gefitinib), and ECOG 1505 (bevacizumab). Clinical cases will be presented to highlight problems and pitfalls of treating patients outside of controlled trials, and new avenues for clinical and translational research will be elucidated. Disclosure The author has declared no conflicts of interest.

What future opportunities may immuno-oncology provide for improving the treatment of patients with lung cancer?

Lung cancer is the leading cause of cancer-related mortality worldwide, with non-small-cell lung cancer (NSCLC) accounting for approximately 85% of all cases. Most patients with NSCLC are diagnosed at an advanced stage and have a poor prognosis, with a 5-year survival rate of <5%. Despite the introduction of new chemotherapeutic agents and molecularly targeted drugs, outcomes remain poor, emphasising the need for new treatment approaches. Inducing or potentiating immune responses via immunotherapeutic manipulation is a viable treatment approach for lung cancer. Antigen-specific, tumour-cell, and dendritic cell-based vaccines have all been evaluated in lung cancer, and some have shown promising clinical activity in phase II trials. These include liposomal BLP25 vaccine (L-BLP25), which targets mucin 1, and melanoma-associated antigen 3 (MAGE-A3) antigen-specific cancer immunotherapeutic (ASCI), which targets MAGE-A3, a peptide expressed almost exclusively on tumour cells. MAGE-A3 ASCI is being evaluated in the adjuvant setting in a phase III trial of patients with early-stage NSCLC, while a phase III trial of L-BLP25 is enrolling patients with unresectable stage III NSCLC. T-cell modulating agents (e.g. antibodies against programmed death 1 and cytotoxic T-lymphocyte-associated antigen-4 [CTLA-4]) are also being investigated. For example, in patients with NSCLC treated with paclitaxel and carboplatin, the phased administration of ipilimumab (an antibody against CTLA-4) resulted in substantial improvements in immune-related progression-free survival compared with chemotherapy alone (5.7 versus 4.6 months; P=0.05). Immunotherapy in lung cancer is starting to deliver promising results in clinical trials. However, further research will be required to establish the optimal timing of therapy (i.e. in the adjuvant or metastatic settings). In addition, it will be important to determine if immunotherapies are most effective when used alone or in combination with other agents.

A phase II trial to assess efficacy and safety of afatinib in extensively pretreated patients with HER2-negative metastatic breast cancer

Afatinib (BIBW 2992) is an ErbB-family blocker that irreversibly inhibits signaling from all relevant ErbB-family dimers. Afatinib has demonstrated preclinical activity in human epidermal growth factor receptor HER2 (ErbB2)-positive and triple-negative xenograft models of breast cancer, and clinical activity in phase I studies. This was a multicenter phase II study enrolling patients with HER2-negative metastatic breast cancer progressing following no more than three lines of chemotherapy. No prior epidermal growth factor receptor-targeted therapy was allowed. Patients received 50-mg afatinib once daily until disease progression. Tumor assessment was performed at every other 28-day treatment course. The primary endpoint was clinical benefit (CB) for ≥4 treatment courses in triple-negative (Cohort A) metastatic breast cancer (TNBC) and objective responses measured by Response Evaluation Criteria in Solid Tumors in patients with HER2-negative, estrogen receptor-positive, and/or progesterone receptor-positive breast cancer (Cohort B). Fifty patients received treatment, including 29 patients in Cohort A and 21 patients in Cohort B. No objective responses were observed in either cohort. Median progression-free survival was 7.4 and 7.7 weeks in Cohorts A and B, respectively. Three patients with TNBC had stable disease for ≥4 treatment courses, one of them for 12 courses (median 26.3 weeks; range 18.9–47.9 weeks). The most frequently observed afatinib-associated adverse events (AEs) were gastrointestinal and skin-related side effects, which were manageable by symptomatic treatment and dose reductions. Afatinib pharmacokinetics were comparable to those observed in previously reported phase I trials. In conclusion, afatinib had limited activity in HER2-negative breast cancer. AEs were generally manageable and mainly affected the skin and the gastrointestinal tract.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-012-2126-1) contains supplementary material, which is available to authorized users.

GAIN-(L): Efficacy and biomarker findings of RG7160 (GA201), a novel, dual-acting monoclonal antibody (mAb) designed to enhance antibody-dependent cellular cytotoxicity (ADCC), in combination with first-line cisplatin and pemetrexed in metastatic nonsquamous NSCLC.

7544 Background: GA201, a humanized, engineered IgG1 anti-Epidermal Growth Factor Receptor (EGFR) mAb designed to enhance ADCC, has shown promising clinical activity in phase I and in the neoadjuvant treatment of head and neck cancer. This phase Ib study (NCT01185847) aimed to determine the safety, pharmacokinetics (PK), activity and recommended phase II dose (RP2D) of GA201 in combination with chemotherapy in non-squamous NSCLC. Methods: Successive cohorts received GA201 1000 mg or 1400 mg (IV d1, d8 then 2-weekly (q2W)) in combination with chemotherapy at standard doses. Data cut off was 7 months after enrolling the last patient. Results: 14 patients (4 female) with performance status 0-1 were enrolled. No maximum tolerated dose was reached. Most common adverse events (AEs – all grades) included rash (100%), hypomagnesaemia (71%), infusion-related reactions (64%), mucosal inflammation (57%) and anemia (50%). AEs of ≥ grade 3 included rash (71%), skin fissure (21%), dry skin (14%), paronychia (14%), and asthenia (14%). Median timeto improvement of rash grade 3 was 11 days. AEs led to dose reduction for 4 patients and discontinuation for 1 patient. There were 6 confirmed partial responses (43%, 5 in 1400 mg cohort) and 7 patients (50%) with stable disease &gt;=9 weeks. Duration of response ranged between 5 and 42 weeks (3 patients still ongoing). Preliminary biomarker analysis shows a correlation between tumor-infiltrating CD16+ immune cells and target lesion shrinkage at first tumor assessment [R(spear)=-0.65 (p=0.02)]; no apparent correlation between EGFR H-score and response was found. PK data supports 1400 mg as the RP2D (d1, d8 then q2W). Conclusions: The RP2D of GA201 in combination with chemotherapy was established to be 1400 mg. The incidence of EGFR associated rash was high and guidelines to reduce its severity were implemented with a noted improvement in tolerability. Promising antitumor activity was observed. Biomarker data support the mode of action of GA201 via ADCC. A randomized phase II trial of this combination is ongoing and fully recruited.

Prolonged Low Dose Therapy with a Pan-Deacetylase Inhibtor, Panobinostat (LBH589), in Patients with Myelofibrosis

Abstract 794▪FN2▪This icon denotes a clinically relevant abstractLBH589 is a novel pan-deacetylase inhibitor (DACi) that has demonstrated clinical activity in phase I/II studies in patients with a variety of hematologic malignancies. Our group has previously presented preliminary results of a phase I study of LBH589 in patients with myelofibrosis (MF) (Mascarenhas et al, ASH 2009, a308) while a phase II trial using higher doses of LBH589 has also been reported (DeAngelo et al, ASH 2010,a630). Both studies identified reversible thrombocytopenia as the DLT and reported evidence of clinical responses. The final results of our phase I study and the effects of extended treatment with LBH589 are reported here. We enrolled 18 patients at 3 dose levels. Fifty-five percent of these patients had PMF, 28% Post-PV MF and 17% Post ET MF; all were intermediate/high risk based on Lille classification. Twenty-five mg PO TIW was determined to be the recommended phase II dose. All patients experienced resolution of their systemic symptoms and 10/11 patients with baseline palpable splenomegaly, who were evaluable after 1 month of therapy, had a median reduction of 30%, range 0–100%. Five patients entered into an extension phase of the trial and received > 6 months of therapy with a mean dose of 20mg PO TIW at time of optimal response (Table 1). Of these patients, 2 were initially enrolled in the 20 mg PO TIW cohort, 1 in the 30 mg PO TIW cohort and 2 in the 25 mg PO TIW cohort. Both patients at the lowest dose achieved clinical improvement (CI) by IWG-MRT response criteria at 6 months as did one patient at the 25 mg dose. The remaining 2 patients had SD at 30 and 25 mg. A mean reduction in palpable splenomegaly at 3 and 6 months of 55% and 83%, respectively, was observed in this group. Two of these patients had marked and durable improvement in anemia (patients 1 and 4). Patient 4 achieved a near CR at 16 months with resolution of palpable splenomegaly, elimination of peripheral blood dacrocytes and leukoerythroblastosis, a 4g/dL increase in hemoglobin, improvement in overall marrow cellularity and megakaryocyte atypia with an increase in erythroid precursors and a significant reduction of reticulin/collagen fibrosis. Patient 1 was heavily transfusion dependent requiring RBC transfusions weekly to maintain a mean hemoglobin of 6.5g/dL and after 6 months on LBH589 achieved >50% reduction in transfusion dependence maintaining a mean hemoglobin of 9g/dL. Patient 2 had resolution of palpable splenomegaly and leukoerythroblastosis by cycle 6 and the bone marrow at cycle 26 was characterized by a reduction in marrow fibrosis from grade 4 to 1. A phase II study is ongoing, 14 patients are currently enrolled, with a planned goal of 22 patients. Pharmacokinetic and pharmacodynamic studies as well as cytokine profiling of the phase I patients are being analyzed and will be presented at the meeting. We conclude that low doses of LBH589 delivered for greater than 6 months in patients with MF are capable of ameliorating symptoms, improving clinical features and reversing pathologic marrow changes. [Display omitted] Disclosures:Off Label Use: Oral histone deacetylase inhibitor that targets epigenetic changes in malignant myelofibrosis cells with an goal to modify the disease process.

Improved Efficacy Upon Combined JAK1/2 and Pan-Deacetylase Inhibition Using Ruxolitinib (INC424) and Panobinostat (LBH589) in Preclinical Mouse Models of JAK2V617F-Driven Disease

Abstract 798▪FN2▪This icon denotes a clinically relevant abstract Background:Ruxolitinib (INC424), a potent and selective oral JAK1 and JAK2 inhibitor, has demonstrated rapid and durable reductions in splenomegaly and improved disease-related symptoms, role functioning, and quality of life in 2 phase 3 studies in patients with myelofibrosis. Panobinostat, a potent oral pan-deacetylase inhibitor (pan-DACi) that increases acetylation of proteins involved in multiple oncogenic pathways, has shown encouraging clinical activity in phase 1/2 trials of myelofibrosis. Here, we investigate the combination of ruxolitinib and panobinostat in mouse models of JAK2V617F-driven disease. Methods:Scid-beige female mice were randomized to treatment groups on the basis of baseline bioluminescence 4 days after injection of Ba/F3 EpoR JAK2V617F-luciferase cells. Mice were treated with vehicle control, panobinostat (4, 8, or 12 mg/kg intraperitoneal 3 times a week) alone or in combination with ruxolitinib (60 mg/kg orally twice daily), or ruxolitinib alone (n=7 each). Whole-body bioluminescence imaging was conducted on days 7 and 11. Spleens were weighed and extracts were obtained to determine levels of phosphorylated STAT5 (p-STAT5) and lysine acetylation. Results:Imaging on day 11 showed luminescence reductions (P <.05 compared with vehicle control) in all treatment groups (Table)Ruxolitinib, mg/kg060000606060Panobino- stat, mg/kg0048124812n=7n=7n=7n=7n=7n=7n=7n=7Change in biolu- mines- cence (T/C),a %10040*27*20*11*,†22*15*,†3*,†‡Spleen weight, mg183.7 ± 18.176.4 ± 17.0*105.3 ± 19.6107.4 ± 20.594.1 ± 17.6*59.9 ± 10.0*45.7 ± 5.6*,‡40.9 ± 6.1*,‡aCalculated as treated/control (T/C) of fractional change in bioluminescence on day 11/day 4*P <.05 vs vehicle control;†P <.05 vs ruxolitinib;‡P <.05 vs panobinostat at the same dose by Tukey’s test on log10 transformed values for fractional change Biolux and spleen weight.A dose-dependent reduction was observed in mice treated with panobinostat alone. Combination therapy produced the greatest reductions in luminescence, as low as 3% of the untreated control in the ruxolitinib + panobinostat (12 mg/kg) group (P <.05 compared with the same dose of panobinostat alone or ruxolitinib alone). Similarly, reductions in spleen weight (P <.05 compared with control) were observed with ruxolitinib alone and panobinostat (12 mg/kg) alone and in all combination groups. Treatment with ruxolitinib reduced phosphorylation of the JAK2 downstream target STAT5, whereas panobinostat treatment reduced total STAT5 levels. Mice treated with both panobinostat and ruxolitinib had reduced levels of p-STAT5 and total STAT5 in spleen extracts. Lysine acetylation, unchanged in ruxolitinib-treated mice, was markedly increased in panobinostat-treated mice. Together, the pharmacodynamic analyses indicate that ruxolitinib and panobinostat are active and have nonoverlapping and complementary effects on biological pathways. There was no major change in tolerability, as assessed by body weight, between panobinostat alone and in combination with ruxolitinib. The experiment was repeated with the 2 top doses of panobinostat and ruxolitinib and showed similar results. Based on these encouraging data, the combination is currently being tested in a mouse JAK2V617F bone marrow transplant model of myeloproliferative neoplasm (MPN) disease to assess its disease-modifying potential. Conclusions:The combination of ruxolitinib and panobinostat showed significant improvements in anticancer activity compared with either treatment given alone in this JAK2V617F mouse model. Further studies in a murine MPN disease model are underway. Disclosures:Baffert:Novartis Pharma AG: Employment. Evrot:Novartis Pharma AG: Employment. Ebel:Novartis Pharma AG: Employment. Roelli:Novartis Pharma AG: Employment. Andraos:Novartis Pharma AG: Employment. Qian:Novartis Pharma AG: Employment. Romanet:Novartis Pharma AG: Employment. Murakami:Novartis Pharma AG: Employment. Radimerski:Novartis Pharma AG: Employment.