Carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) in Pancreatic Ductal Adenocarcinoma (PDA): An integrative analysis of a novel therapeutic target.

We investigated biomarker CEACAM6, a highly abundant cell surface adhesion receptor that modulates the extracellular matrix (ECM) in pancreatic ductal adenocarcinoma (PDA). The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) RNA-Seq data from PDA patients were analyzed for CEACAM6 expression and evaluated for overall survival, association, enrichment and correlations. A CRISPR/Cas9 Knockout (KO) of CEACAM6 in PDA cell line for quantitative proteomics, mitochondrial bioenergetics and tumor growth in mice were conducted. We found CEACAM6 is over-expressed in primary and metastatic basal and classical PDA subtypes. Highest levels are in classical activated stroma subtype. CEACAM6 over-expression is universally a poor prognostic marker in KRAS mutant and wild type PDA. High CEACAM6 expression is associated with low cytolytic T-cell activity in both basal and classical PDA subtypes and correlates with low levels of T-REG markers. In HPAF-II cells knockout of CEACAM6 alters ECM-cell adhesion, catabolism, immune environment, transmembrane transport and autophagy. CEACAM6 loss increases mitochondrial basal and maximal respiratory capacity. HPAF-II CEACAM6-/- cells are growth suppressed by >65% vs. wild type in mice bearing tumors. CEACAM6, a key regulator affects several hallmarks of PDA including the fibrotic reaction, immune regulation, energy metabolism and is a novel therapeutic target in PDA.

Ublituximab for the treatment of CD20 positive B-cell malignancies.

INTRODUCTION: Non-Hodgkin lymphoma (NHL) is the most common adult hematologic malignancy. Conventional methods of treatment are chemotherapy and radiation, which were associated with toxicities and lack of specificity. Potential cell surface targets for treatment of B-cell NHL (B-NHL) include CD19, CD20, and CD22 which are highly expressed on malignant B-cells. The development of monoclonal antibody (mAb) therapy directed against CD20 had the most clinical impact in the treatment of B-NHL. Early clinical trials with rituximab (RTX), the first chimeric mAb against CD20, showed efficacy and minimal toxicities. RTX was later approved as first line in combination with CHOP chemotherapy for Diffuse Large B-NHL (DLBCL). The emergence of resistance to RTX prompted the development of the next-generation of mAbs targeting CD20 (e.g. obinituzumab, ofatumumab), and includes ublituximab (Ub), with higher complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) against malignant B-cells. Areas covered: Herein, we discuss clinical trials of Ub, highlighting efficacy, tolerability and an expert opinion on drug development in B-NHL. A pubmed search was conducted to evaluate all Ub clinical trials. Expert opinion: Ub demonstrated efficacy in patients with high-risk CLL and B-NHL in both first line, subsequent lines, and in rituximab refractory patients.

Therapeutic potential of investigational CHK-1 inhibitors for the treatment of solid tumors.

INTRODUCTION: For several decades' cancer treatment targeting DNA repair pathways incorporated both chemo- and radiotherapy only. However, over the last decade improved knowledge of DNA repair processes has paved the way for the development of novel targeted drugs abrogating DNA repair signaling. Checkpoint kinase inhibitors are exciting molecules and hold promise in the treatment of both solid and hematologic malignancies. Herein, we discuss preclinical and clinical studies with this class of molecules. Areas covered: In this review, we discuss the role of check point kinase 1 (CHK-1) in DNA repair and provide a comprehensive summary of pre-clinical and early phase clinical trials with CHK-1 inhibitors. We also provide molecular structural basis of CHK-1inhibitors binding to CHK-1. Expert opinion: Available data from both pre-clinical and early clinical studies illustrates potential efficacy of this class of molecules when combined with antimetabolites in treating both solid and hematologic malignancies. In addition, there might be an additive role in combining this class of molecules to PARP inhibitors, platinum chemotherapy, or radiation therapy in p53 or BRCA mutated tumors. The safety of the aforementioned combination needs to be closely evaluated in the ongoing clinical trials.

A novel small molecule inhibitor of the DNA repair protein Ku70/80.

Non-Homologous End-Joining (NHEJ) is the predominant pathway for the repair of DNA double strand breaks (DSBs) in human cells. The NHEJ pathway is frequently upregulated in several solid cancers as a compensatory mechanism for a separate DSB repair defect or for innate genomic instability, making this pathway a powerful target for synthetic lethality approaches. In addition, NHEJ reduces the efficacy of cancer treatment modalities which rely on the introduction of DSBs, like radiation therapy or genotoxic chemotherapy. Consequently, inhibition of the NHEJ pathway can modulate a radiation- or chemo-refractory disease presentation. The Ku70/80 heterodimer protein plays a pivotal role in the NHEJ process. It possesses a ring-shaped structure with high affinity for DSBs and serves as the first responder and central scaffold around which the rest of the repair complex is assembled. Because of this central position, the Ku70/80 dimer is a logical target for the disruption of the entire NHEJ pathway. Surprisingly, specific inhibitors of the Ku70/80 heterodimer are currently not available. We here describe an in silico, pocket-based drug discovery methodology utilizing the crystal structure of the Ku70/80 heterodimer. We identified a novel putative small molecule binding pocket and selected several potential inhibitors by computational screening. Subsequent biological screening resulted in the first identification of a compound with confirmed Ku-inhibitory activity in the low micro-molar range, capable of disrupting the binding of Ku70/80 to DNA substrates and impairing Ku-dependent activation of another NHEJ factor, the DNA-PKCS kinase. Importantly, this compound synergistically sensitized human cell lines to radiation treatment, indicating a clear potential to diminish DSB repair. The chemical scaffold we here describe can be utilized as a lead-generating platform for the design and development of a novel class of anti-cancer agents.

Reciprocal feedback inhibition of the androgen receptor and PI3K as a novel therapy for castrate-sensitive and -resistant prostate cancer.

Gain-of-function of the androgen receptor (AR) and activation of PI3K/AKT/mTOR pathway have been demonstrated to correlate with progression to castration-resistant prostate cancer (CRPC). However, inhibition of AR or PI3K/mTOR alone results in a reciprocal feedback activation. Therefore, we hypothesized that dual inhibition of the AR and PI3K/mTOR pathway might lead to a synergistic inhibition of cell growth and overcome drug resistance in CRPC. Here, we reported that androgen-depletion increased AR protein level and Akt phosphorylation at Ser473 and Thr308 in LNCaP cells. Moreover, we developed resistance cell lines of LNCaP to Enzalutamide (or MDV3100), an AR inhibitor (named as LNCaP 'MDV-R') and PF-04691502, a PI3K/mTOR inhibitor (named as LNCaP 'PF-R'). MTS analysis showed that LNCaP 'PF-R' was strongly resistant to Enzalutamide treatment, and on the other hand, LNCaP 'MDV-R' was 6-fold resistant to PF-04691502 treatment. Mechanistically, LNCaP 'MDV-R' cells had significantly reduced AR, loss of PSA and increase Akt activity in contrast with LNCaP 'PF-R' cells. Combined inhibition of PI3K/mTOR and AR pathways with a variety of small molecular inhibitors led to a synergistic suppression of cell proliferation and a profound increase of apoptosis and cell cycle arrest in both androgen-dependent LNCaP and independent CRPC 22Rv1 cell lines. In conclusion, this study provides preclinical proof-of-concept that the combination of a PI3K/mTOR inhibitor with an AR inhibitor results in a synergistic anti-tumor response in non-CRPC and CRPC models.

Novel receptor tyrosine kinase targeted combination therapies for imatinib-resistant gastrointestinal stromal tumors (GIST).

BACKGROUND: c-Kit/α-PDGFR targeted therapies are effective for gastrointestinal stromal tumors (GIST), but, >50% develop drug resistance. METHODS: RTK expression (c-Kit, c-Met, AXL, HER-1, HER-2, IGF-1R) in pre-/post-imatinib (IM) GIST patient samples (n=16) and 4 GIST cell lines were examined for RTK inhibitor activity. GIST-882 cells were cultured in IM every other day, cells collected (1 week to 6 months) and analyzed by qRT-PCR and Western blotting. RESULTS: Immunohistochemistry pre-/post-IM demonstrated continued expression of c-Kit and HER1, while a subset expressed IGF-1R, c-Met and AXL. In GIST cells (GIST-882, GIST430/654, GIST48) c-Kit, HER1 and c-Met are co-expressed. Acute IM over-express c-Kit while chronic IM, lose c-Kit and HER-1 in GIST882 cells. GIST882 and GIST430/654 cells have an IC50 0.077 and 0.59 µM to IM respectively. GIST48 have an IC50 0.66 µM to IM, 0.91 µM to amuvatinib [AMU] and 0.67 µM to erlotinib (Erl). Synergistic combinations: GIST882, AMU + Erl (CI 0.20); IM + AMU (CI 0.50), GIST430/654, IM + afatinib (CI 0.39); IM + AMU (CI 0.42), GIST48, IM + afatinib (CI 0.03); IM + AMU (CI 0.04); AMU + afatinib (CI 0.36); IM + Erl (CI 0.63). CONCLUSION: Targeting c-Kit plus HER1 or AXL/c-Met abrogates IM resistance in GIST.

Alisertib added to rituximab and vincristine is synthetic lethal and potentially curative in mice with aggressive DLBCL co-overexpressing MYC and BCL2.

Pearson correlation coefficient for expression analysis of the Lymphoma/Leukemia Molecular Profiling Project (LLMPP) demonstrated Aurora A and B are highly correlated with MYC in DLBCL and mantle cell lymphoma (MCL), while both Auroras correlate with BCL2 only in DLBCL. Auroras are up-regulated by MYC dysregulation with associated aneuploidy and resistance to microtubule targeted agents such as vincristine. Myc and Bcl2 are differentially expressed in U-2932, TMD-8, OCI-Ly10 and Granta-519, but only U-2932 cells over-express mutated p53. Alisertib [MLN8237 or M], a highly selective small molecule inhibitor of Aurora A kinase, was synergistic with vincristine [VCR] and rituximab [R] for inhibition of cell proliferation, abrogation of cell cycle checkpoints and enhanced apoptosis versus single agent or doublet therapy. A DLBCL (U-2932) mouse model showed tumor growth inhibition (TGI) of ∼ 10-20% (p = 0.001) for M, VCR and M-VCR respectively, while R alone showed ∼ 50% TGI (p = 0.001). M-R and VCR-R led to tumor regression [TR], but relapsed 10 days after discontinuing therapy. In contrast, M-VCR-R demonstrated TR with no relapse >40 days after stopping therapy with a Kaplan-Meier survival of 100%. Genes that are modulated by M-VCR-R (CENP-C, Auroras) play a role in centromere-kinetochore function in an attempt to maintain mitosis in the presence of synthetic lethality. Together, our data suggest that the interaction between alisertib plus VCR plus rituximab is synergistic and synthetic lethal in Myc and Bcl-2 co-expressing DLBCL. Alisertib plus vincristine plus rituximab [M-VCR-R] may represent a new strategy for DLBCL therapy.

Alisertib (MLN8237) an investigational agent suppresses Aurora A and B activity, inhibits proliferation, promotes endo-reduplication and induces apoptosis in T-NHL cell lines supporting its importance in PTCL treatment.

Peripheral T-cell lymphomas (PTCL) are a diverse group of rare non-Hodgkin lymphomas (NHL) that carry a poor prognosis and are in need of effective therapies. Alisertib (MLN8237) an investigational agent that inhibits Aurora A Ser/Thr kinase has shown activity in PTCL patients. Here we demonstrate that aurora A and B are highly expressed in T-cell lymphoma cell lines. In PTCL patient samples aurora A was positive in 3 of 24 samples and co-expressed with aurora B. Aurora B was positive in tumor cells in 22 of 32 samples. Of the subtypes of PTCL, aurora B was over-expressed in PTCL (NOS) [73%], T-NHL [100%], ALCL (Alk-Neg) [100%] and AITL [100%]. Treatment with MLN8237 inhibited PTCL cell proliferation in CRL-2396 and TIB-48 cells with an IC50 of 80-100nM. MLN8237 induced endo-reduplication in a dose and time dependent manner in PTCL cell lines leading to apoptosis demonstrated by flow cytometry and PARP-cleavage at concentrations achieved in early phase clinical trials. Moreover, inhibition of HisH3 and aurora A phosphorylation was dose dependent and strongly correlated with endo-reduplication. The data provide a sound rationale for aurora inhibition in PTCL as a therapeutic modality and warrants clinical trial evaluation.

Aurora A inhibitor (MLN8237) plus vincristine plus rituximab is synthetic lethal and a potential curative therapy in aggressive B-cell non-Hodgkin lymphoma.

PURPOSE: Aurora A and B are oncogenic serine/threonine kinases that regulate mitosis. Overexpression of Auroras promotes resistance to microtubule-targeted agents. We investigated mechanistic synergy by inhibiting the mitotic spindle apparatus in the presence of MLN8237 [M], an Aurora A inhibitor with either vincristine [MV] or docetaxel [MD] in aggressive B-cell non-Hodgkin lymphoma (B-NHL). The addition of rituximab [R] to MV or MD was evaluated for synthetic lethality. EXPERIMENTAL DESIGN: Aggressive B-NHL cell subtypes were evaluated in vitro and in vivo for target modulation and anti-NHL activity with single agents, doublets, and triplets by analyzing cell proliferation, apoptosis, tumor growth, survival, and mechanisms of response/relapse by gene expression profiling with protein validation. RESULTS: MV is synergistic whereas MD is additive for cell proliferation inhibition in B-NHL cell culture models. Addition of rituximab to MV is superior to MD, but both significantly induce apoptosis compared with doublet therapy. Mouse xenograft models of mantle cell lymphoma showed modest single-agent activity for MLN8237, rituximab, docetaxel, and vincristine with tumor growth inhibition (TGI) of approximately 10% to 15%. Of the doublets, MV caused tumor regression, whereas TGI was observed with MD (approximately 55%-60%) and MR (approximately 25%-50%), respectively. Although MV caused tumor regression, mice relapsed 20 days after stopping therapy. In contrast, MVR was curative, whereas MDR led to TGI of approximately 85%. Proliferation cell nuclear antigen, Aurora B, cyclin B1, cyclin D1, and Bcl-2 proteins of harvested tumors confirmed response and resistance to therapy. CONCLUSIONS: Addition of rituximab to MV is a novel therapeutic strategy for aggressive B-NHL and warrants clinical trial evaluation.

AT9283, a novel aurora kinase inhibitor, suppresses tumor growth in aggressive B-cell lymphomas.

Aurora kinases are oncogenic serine/threonine kinases that play key roles in regulating the mitotic phase of the eukaryotic cell cycle. Auroras are overexpressed in numerous tumors including B-cell non-Hodgkin's lymphomas and are validated oncology targets. AT9283, a pan-aurora inhibitor inhibited growth and survival of multiple solid tumors in vitro and in vivo. In this study, we demonstrated that AT9283 had potent activity against Aurora B in a variety of aggressive B-(non-Hodgkin lymphoma) B-NHL cell lines. Cells treated with AT9283 exhibited endoreduplication confirming the mechanism of action of an Aurora B inhibitor. Also, treatment of B-NHL cell lines with AT9283 induced apoptosis in a dose and time dependent manner and inhibited cell proliferation with an IC(50) < 1 µM. It is well known that inhibition of auroras (A or B) synergistically enhances the effects of microtubule targeting agents such as taxanes and vinca alkaloids to induce antiproliferation and apoptosis. We evaluated whether AT9283 in combination with docetaxel is more efficient in inducing apoptosis than AT9283 or docetaxel alone. At very low doses (5 nM) apoptosis was doubled in the combination (23%) compared to AT9283 or docetaxel alone (10%). A mouse xenograft model of mantle cell lymphoma demonstrated that AT9283 at 15 mg/kg and docetaxel (10 mg/kg) alone had modest anti-tumor activity. However, AT9283 at 20 mg/kg and AT9283 (15 or 20 mg/kg) plus docetaxel (10 mg/kg) demonstrated a statistically significant tumor growth inhibition and enhanced survival. Together, our results suggest that AT9283 plus docetaxel may represent a novel therapeutic strategy in B-cell NHL and warrant early phase clinical trial evaluation.

An analog of withaferin A activates the MAPK and glutathione "stress" pathways and inhibits pancreatic cancer cell proliferation.

Withaferin A (WA) (1) and two analogs [4-epi-withaferin A (2) and 4,27-diacetyl-4-epi-withaferin A (3)] were evaluated for antitumor activity in pancreatic cancer cells. IC(50) for 1, 2, and 3 were 0.87, 0.45, and 0.29 ?M (BxPC-3); 1.28, 1.53, and 0.52 ?M (MIAPaCa-2); and 0.59, 2.25, and 0.56 ?M (PANC-1), respectively. We chose WA analog 3 for functional studies with confirmatory RT-PCR and Western blotting. ANOVA identified 33 (MIAPaCa-2), 54 (PANC-1), and 48 (BxPC-3) gene expression changes. Fisher exact test demonstrated MAPK and glutathione pathways to be overexpressed with WA analog 3. WA analog 3 elicits a dose- and time-dependent apoptosis, activates MAPK and glutathione ?stress? pathways, and inhibits proliferation.

Aurora inhibitor MLN8237 in combination with docetaxel enhances apoptosis and anti-tumor activity in mantle cell lymphoma.

Auroras (A and B) are oncogenic serine/threonine kinases that play key roles in the mitotic phase of the eukaryotic cell cycle. Analysis of the leukemia lymphoma molecular profiling project (LLMPP) database indicates Aurora over-expression correlates with poor prognosis. A tissue microarray (TMA) composed of 20 paired mantle cell lymphoma (MCL) patients demonstrated >75% of patients had high levels Aurora expression. Aurora A and B were also found elevated in 13 aggressive B-NHL cell lines. MLN8237, an Aurora inhibitor induced G2/M arrest with polyploidy and abrogated Aurora A and histone-H3 phosphorylation. MLN8237 inhibited aggressive B-NHL cell proliferation at an IC(50) of 10-50 nM and induced apoptosis in a dose- and time-dependent manner. Low dose combinations of MLN8237+docetaxel enhanced apoptosis by ~3-4-fold in cell culture compared to single agents respectively. A mouse xenograft model of MCL demonstrated that MLN8237 (10 or 30 mg/kg) or docetaxel (10mg/kg) alone had modest anti-tumor activity. However, MLN8237 plus docetaxel demonstrated a statistically significant tumor growth inhibition and enhanced survival compared to single agent therapy. Together, our results suggest that MLN8237 plus docetaxel may represent a novel therapeutic strategy that could be evaluated in early phase trials in relapsed/refractory aggressive B-cell NHL.

Design and activity of a murine and humanized anti-CEACAM6 single-chain variable fragment in the treatment of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease, with surgery being the only curative modality for localized disease, and gemcitabine with or without erlotinib remains the standard of therapy for unresectable or metastatic disease. CEACAM6 is overexpressed in human PDA independent of stage or grade and causes anoikis resistance when dysregulated. Because murine monoclonal antibody 13-1 possesses target-specific cytotoxicity in human PDA cell lines, we designed a humanized anti-CEACAM6 single-chain variable fragment (scFv) based on monoclonal antibody 13-1. PEGylation of the glycine-serine linker was used to enhance plasma half-life. These scFvs bound CEACAM6 with high affinity, exhibited cytotoxic activity, and induced dose-dependent poly(ADP-ribose) polymerase cleavage. Murine PDA xenograft models treated with humanized scFv alone elicited tumor growth inhibition, which was enhanced in combination with gemcitabine. Immunohistochemistry showed significant apoptosis, with inhibition of angiogenesis and proliferation, and preservation of the target. Collectively, our results have important implications for the development of novel antibody-based therapies against CEACAM6 in PDA.