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In vivo Efficacy of ABBV-321 tumor targeting.

ABBV-321 (serclutamab talirine), a next-generation EGFR-targeted antibody–drug conjugate (ADC) incorporates a potent pyrrolobenzodiazepine (PBD) dimer toxin conjugated to the EGFR-targeting ABT-806 affinity-matured AM1 antibody. ABBV-321 follows the development of related EGFR-targeted ADCs including depatuxizumab mafodotin (depatux-m, ABT-414), ABT-806 conjugated to monomethyl auristatin F (MMAF), and ABBV-221 (losatuxizumab vedotin), AM1 antibody conjugated to monomethyl auristatin E (MMAE). The distinct tumor selectivity of ABBV-321 differentiates it from many previous highly active antibody PBD conjugates that lack a therapeutic window. Potency of the PBD dimer, combined with increased binding of AM1 to EGFR-positive tumor cells, opens the possibility to target a wide array of tumors beyond those with high levels of EGFR overexpression or amplification, including those insensitive to auristatin-based ADCs. ABBV-321 exhibits potent antitumor activity in cellular and in vivo studies including xenograft cell line and patient-derived xenograft glioblastoma, colorectal, lung, head and neck, and malignant mesothelioma tumor models that are less sensitive to depatux-m or ABBV-221. Combination studies with ABBV-321 and depatux-m suggest a promising treatment option permitting suboptimal, and potentially better tolerated, doses of both ADCs while providing improved potency. Collectively, these data suggest that ABBV-321 may offer an extended breadth of efficacy relative to other EGFR ADCs while extending utility to multiple EGFR-expressing tumor indications. Despite its highly potent PBD dimer payload, the tumor selectivity of ABBV-321, coupled with its pharmacology, toxicology, and pharmacokinetic profiles, support continuation of ongoing phase I clinical trials in patients with advanced EGFR-expressing malignancies.

Cross species binding and tolerability of ABBV-085.

Figures S1-S7 show LRRC15 RNA and protein expression in cancer, and demonstrate the anti-tumor efficacy of the LRRC15-targeted ADC ABBV-085 in vitro and in vivo as a monotherapy and in combination with standard-of-care therapies.

Supplementary Figure Legends

Progress in understanding tumor stromal biology has been constrained in part because cancer-associated fibroblasts (CAF) are a heterogeneous population with limited cell-type–specific protein markers. Using RNA expression profiling, we identified the membrane protein leucine-rich repeat containing 15 (LRRC15) as highly expressed in multiple solid tumor indications with limited normal tissue expression. LRRC15 was expressed on stromal fibroblasts in many solid tumors (e.g., breast, head and neck, lung, pancreatic) as well as directly on a subset of cancer cells of mesenchymal origin (e.g., sarcoma, melanoma, glioblastoma). LRRC15 expression was induced by TGFβ on activated fibroblasts (αSMA+) and on mesenchymal stem cells. These collective findings suggested LRRC15 as a novel CAF and mesenchymal marker with utility as a therapeutic target for the treatment of cancers with LRRC15-positive stromal desmoplasia or cancers of mesenchymal origin. ABBV-085 is a monomethyl auristatin E (MMAE)-containing antibody–drug conjugate (ADC) directed against LRRC15, and it demonstrated robust preclinical efficacy against LRRC15 stromal-positive/cancer-negative, and LRRC15 cancer-positive models as a monotherapy, or in combination with standard-of-care therapies. ABBV-085′s unique mechanism of action relied upon the cell-permeable properties of MMAE to preferentially kill cancer cells over LRRC15-positive CAF while also increasing immune infiltrate (e.g., F4/80+ macrophages) in the tumor microenvironment. In summary, these findings validate LRRC15 as a novel therapeutic target in multiple solid tumor indications and support the ongoing clinical development of the LRRC15-targeted ADC ABBV-085.Significance: These findings identify LRRC15 as a new marker of cancer-associated fibroblasts and cancers of mesenchymal origin and provide preclinical evidence for the efficacy of an antibody-drug conjugate targeting the tumor stroma. Cancer Res; 78(14); 4059–72. ©2018 AACR.

Purpose: Despite the importance of the MET oncogene in many malignancies, clinical strategies targeting c-Met have benefitted only small subsets of patients with tumors driven by signaling through the c-Met pathway, thereby necessitating selection of patients with MET amplification and/or c-Met activation most likely to respond. An ADC targeting c-Met could overcome these limitations with potential as a broad-acting therapeutic.Experimental Design: ADC ABBV-399 was generated with the c-Met–targeting antibody, ABT-700. Antitumor activity was evaluated in cancer cells with overexpressed c-Met or amplified MET and in xenografts including patient-derived xenograft (PDX) models and those refractory to other c-Met inhibitors. The correlation between c-Met expression and sensitivity to ABBV-399 in tumor and normal cell lines was assessed to evaluate the risk of on-target toxicity.Results: A threshold level of c-Met expressed by sensitive tumor but not normal cells is required for significant ABBV-399–mediated killing of tumor cells. Activity extends to c-Met or amplified MET cell line and PDX models where significant tumor growth inhibition and regressions are observed. ABBV-399 inhibits growth of xenograft tumors refractory to other c-Met inhibitors and provides significant therapeutic benefit in combination with standard-of-care chemotherapy.Conclusions: ABBV-399 represents a novel therapeutic strategy to deliver a potent cytotoxin to c-Met–overexpressing tumor cells enabling cell killing regardless of reliance on MET signaling. ABBV-399 has progressed to a phase I study where it has been well tolerated and has produced objective responses in c-Met–expressing non–small cell lung cancer (NSCLC) patients. Clin Cancer Res; 23(4); 992–1000. ©2016 AACR.

ABBV-399, a c-Met Antibody Drug Conjugate that Targets Both MET Amplified and c-Met Overexpressing Tumors, Irrespective of MET Pathway Dependence. Figure S1 shows ABBV-399 is internalized upon binding to tumor cells. Figure S2 shows the down-regulation of phospho- and total c-Met and downstream signaling molecules upon treatment with ABBV-399

Supplementary Data from ABT-888 Confers Broad In vivo Activity in Combination with Temozolomide in Diverse Tumors

Purpose: ABT-888, currently in phase 2 trials, is a potent oral poly(ADP-ribose) polymerase inhibitor that enhances the activity of multiple DNA-damaging agents, including temozolomide (TMZ). We investigated ABT-888+TMZ combination therapy in multiple xenograft models representing various human tumors having different responses to TMZ.Experimental Design: ABT-888+TMZ efficacy in xenograft tumors implanted in subcutaneous, orthotopic, and metastatic sites was assessed by tumor burden, expression of poly(ADP-ribose) polymer, and O6-methylguanine methyltransferase (MGMT).Results: Varying levels of ABT-888+TMZ sensitivity were evident across a broad histologic spectrum of models (55-100% tumor growth inhibition) in B-cell lymphoma, small cell lung carcinoma, non–small cell lung carcinoma, pancreatic, ovarian, breast, and prostate xenografts, including numerous regressions. Combination efficacy in otherwise TMZ nonresponsive tumors suggests that TMZ resistance may be overcome by poly(ADP-ribose) polymerase inhibition. Profound ABT-888+TMZ efficacy was seen in experimental metastases models that acquired resistance to TMZ. Moreover, TMZ resistance was overcome in crossover treatments, indicating that combination therapy may overcome acquired TMZ resistance. Neither tumor MGMT, mismatch repair, nor poly(ADP-ribose) polymer correlated with the degree of sensitivity to ABT-888+TMZ.Conclusions: Robust ABT-888+TMZ efficacy is observed across a spectrum of tumor types, including orthotopic and metastatic implantation. As many TMZ nonresponsive tumors proved sensitive to ABT-888+TMZ, this novel combination may broaden the clinical use of TMZ beyond melanoma and glioma. Although TMZ resistance may be influenced by MGMT, neither MGMT nor other mechanisms of TMZ resistance (mismatch repair) precluded sensitivity to ABT-888+TMZ. Underlying mechanisms of TMZ resistance in these models are not completely understood but likely involve mechanisms independent of MGMT.(Clin Cancer Res 2009;15(23):7277–90)

Purpose: Depatux-m is an antibody drug conjugate (ADC) that targets and inhibits growth of cancer cells overexpressing the epidermal growth factor receptor (EGFR) or the 2–7 deletion mutant (EGFRvIII) in tumor models in vitro and in vivo. Treatment of patients suffering from relapsed/refractory glioblastoma (GBM) with a combination of depatux-m and temozolomide (TMZ) tended to increase overall survival. As a first step to understand the nature of the interaction between the two drugs, we investigated whether the interaction was synergistic, additive or antagonistic. Methods: The efficacy of ADCs, antibodies, TMZ and radiation was tested in xenograft models of GBM, U-87MG and U-87MG EGFRvIII. Both models express EGFR. U-87MG EGFRvIII was transduced to express EGFRvIII. Changes in tumor volume, biomarkers of cell death and apoptosis after treatment were used to measure efficacy of the various treatments. Synergism of depatux-m and TMZ was verified in three-dimensional cultures of U-87MG and U-87MG EGFRvIII by the method of Chou and Talalay. Results: Combined with TMZ and radiotherapy (RT), depatux-m inhibited xenograft growth of U-87MG and U-87MG EGFRvIII more than either treatment with depatux-m or TMZ + RT. Durability of the response to depatux-m + TMZ + RT or depatux-m + TMZ was more pronounced in U-87MG EGFRvIII than in U-87MG. Efficacy of depatux-m + TMZ was synergistic in U-87MG EGFRvIII and additive in U-87MG. Conclusion: Adding depatux-m enhances the efficacy of standard of care therapy in preclinical models of GBM. Durability of response to depatux-m + TMZ in vivo and synergy of the drug-drug interaction correlates with the amount of antigen expressed by the tumor cells.

ABBV-321 (serclutamab talirine), a next-generation EGFR-targeted antibody–drug conjugate (ADC) incorporates a potent pyrrolobenzodiazepine (PBD) dimer toxin conjugated to the EGFR-targeting ABT-806 affinity-matured AM1 antibody. ABBV-321 follows the development of related EGFR-targeted ADCs including depatuxizumab mafodotin (depatux-m, ABT-414), ABT-806 conjugated to monomethyl auristatin F (MMAF), and ABBV-221 (losatuxizumab vedotin), AM1 antibody conjugated to monomethyl auristatin E (MMAE). The distinct tumor selectivity of ABBV-321 differentiates it from many previous highly active antibody PBD conjugates that lack a therapeutic window. Potency of the PBD dimer, combined with increased binding of AM1 to EGFR-positive tumor cells, opens the possibility to target a wide array of tumors beyond those with high levels of EGFR overexpression or amplification, including those insensitive to auristatin-based ADCs. ABBV-321 exhibits potent antitumor activity in cellular and in vivo studies including xenograft cell line and patient-derived xenograft glioblastoma, colorectal, lung, head and neck, and malignant mesothelioma tumor models that are less sensitive to depatux-m or ABBV-221. Combination studies with ABBV-321 and depatux-m suggest a promising treatment option permitting suboptimal, and potentially better tolerated, doses of both ADCs while providing improved potency. Collectively, these data suggest that ABBV-321 may offer an extended breadth of efficacy relative to other EGFR ADCs while extending utility to multiple EGFR-expressing tumor indications. Despite its highly potent PBD dimer payload, the tumor selectivity of ABBV-321, coupled with its pharmacology, toxicology, and pharmacokinetic profiles, support continuation of ongoing phase I clinical trials in patients with advanced EGFR-expressing malignancies.

Purpose: Despite the importance of the MET oncogene in many malignancies, clinical strategies targeting c-Met have benefitted only small subsets of patients with tumors driven by signaling through the c-Met pathway, thereby necessitating selection of patients with MET amplification and/or c-Met activation most likely to respond. An ADC targeting c-Met could overcome these limitations with potential as a broad-acting therapeutic. Experimental Design: ADC ABBV-399 was generated with the c-Met–targeting antibody, ABT-700. Antitumor activity was evaluated in cancer cells with overexpressed c-Met or amplified MET and in xenografts including patient-derived xenograft (PDX) models and those refractory to other c-Met inhibitors. The correlation between c-Met expression and sensitivity to ABBV-399 in tumor and normal cell lines was assessed to evaluate the risk of on-target toxicity. Results: A threshold level of c-Met expressed by sensitive tumor but not normal cells is required for significant ABBV-399–mediated killing of tumor cells. Activity extends to c-Met or amplified MET cell line and PDX models where significant tumor growth inhibition and regressions are observed. ABBV-399 inhibits growth of xenograft tumors refractory to other c-Met inhibitors and provides significant therapeutic benefit in combination with standard-of-care chemotherapy. Conclusions: ABBV-399 represents a novel therapeutic strategy to deliver a potent cytotoxin to c-Met–overexpressing tumor cells enabling cell killing regardless of reliance on MET signaling. ABBV-399 has progressed to a phase I study where it has been well tolerated and has produced objective responses in c-Met–expressing non–small cell lung cancer (NSCLC) patients. Clin Cancer Res; 23(4); 992–1000. ©2016 AACR.

ABT-700 is a therapeutic antibody against the hepatocyte growth factor receptor (MET). At doses or regimens that lead to exposures exceeding optimum in vivo, the efficacy of ABT-700 is unexpectedly reduced. We hypothesized that this reduction in efficacy was due to a “prozone-like” effect in vivo. A prozone-like effect, which is a reduction in efficacy beyond optimum exposure, is caused due a mechanism similar to the generation of false negative flocculation tests by excessive antibody titres. In vitro, we demonstrate that at higher ABT-700 concentrations, this “prozone-like” effect is mediated by a progressive conversion from bivalent to ineffective monovalent binding of the antibody. In vivo, the efficacy of ABT-700 is dependent on an optimum range of exposure as well. Our data suggest that the “prozone-like” effect is operative and independent of target expression. ABT-700 dose, regimen, exposure, and tumor burden are interdependent variables influencing the “prozone-like” effect and mediating and in vivo efficacy. By optimization of dosage and regimen we demonstrate that the “prozone-like” effect can be alleviated and ABT-700 efficacy at varying tumor loads can be further extended in combination with cisplatin. Our results suggest that optimization of exposure taking tumor burden into account may alleviate “prozone-like” effects without compromising efficacy.

e15668 Background: Eftozanermin alfa (eftoza; formerly known as ABBV-621), a 2nd-generation tumor necrosis factor-related apoptosis-inducing ligand receptor agonist, is being evaluated in previously treated solid and hematologic malignancies (NCT03082209). In a dose-expansion cohort, patients (pts) with KRAS-mutant colorectal cancer (n = 24) and pancreatic cancer (n = 24) were evaluated at 3 dose levels with 12 mandatory paired biopsies per tumor type (pretreatment [Tx] and on-Tx collection). Following eftoza dosing, RNA and protein expression including posttranslational modifications were assessed in tumor biopsies to understand the target engagement and downstream pathway activation. Plasma was evaluated for changes in somatic mutant allele frequency and M30, M65 (circulating apoptotic markers). Methods: Biopsies were collected anytime during the screening period (pre-Tx) and 24±4 h following 2nd or 3rd infusion (on-Tx). Of the requested 4–6 fresh biopsy cores, 1–2 cores were collected as formalin fixed paraffin embedded (FFPE) and the rest were frozen tissue. FFPE tissue was analyzed by multiplex immunohistochemistry (IHC) and RNAseq; reverse phase protein array was used for frozen cores. Plasma was collected at cycle 1 predose and 2, 8, 24, 48, and 168 h postdose and analyzed for M30, M65 (by ELISA) and circulating tumor DNA (64-gene PlasmaSELECT assay). Results: Twenty-five pts consented to biopsies; paired biopsies were obtained from 16 pts at a 64% success rate: FFPE (n = 15) and frozen cores (n = 12). Tumor cells were detected in 11/15 (73%) FFPE and 4/12 (33%) frozen cores. Increase in M30, activated caspases, and cleaved PARP levels was observed in on-Tx biopsy samples compared with pre-Tx, thus serving as evidence for apoptosis induction in tumors following eftoza dosing. Changes in the tumor microenvironment were observed post-Tx by RNAseq and multiplex IHC (eg, CD68 level). Downregulation of prosurvival signaling pathways (eg, AKT/MEK) was also observed following eftoza dosing. Thirteen out of 16 pts showed transient increase in mutant allele fractions post eftoza Tx that correlated with increased plasma circulating tumor markers M30 and M65 at similar time points, suggesting activation of apoptosis pathway. Increase in M30, M65 levels also preceded increase in liver enzymes (ALT/AST) at 2, 48 hr post-Tx. Conclusions: Pharmacodynamic effect of eftoza was successfully demonstrated in blood and tumor tissue, including induction of apoptosis and modulation of PI3K and MEK signaling pathways. Clinical trial information: NCT03082209.

Understanding the cell types present in the tumor microenvironment and their activation state is at the forefront of immunotherapy research. To address this, MIBI has been developed to simultaneously image more than 40 markers at single cell resolution. Staining of 10 NSCLC formalin-fixed paraffin embedded tissue sections was performed with a panel of 20 metal labeled antibodies stained together. The tissue was imaged at subcellular resolution using an ion beam and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Masses of detected species were assigned to target biomolecules given the unique label of each antibody and multi-step processing and segmentation were performed to create images of the TME. Each tumor sample was imaged across 10 regions of interest (ROIs) to assess heterogeneity of the TME. The resulting single cell segmentation enabled quantitative analyses of both marker expression and the spatial relationships between cells of different types. At the highest level, cells were classified as immune (CD45) and tumor cells (keratin) based on measured intensities of marker expression. Co-expression of markers were used to classify B cells, macrophages, and T cells subsets. Markers associated with immune suppression such as PD-L1 were also quantified. Cell types and their frequency were compared within the 10 ROIs collected per sample as well as between samples. Finally, distances between tumor and the closest immune cell were measured as a means for describing the spatial organization of the TME, which has been linked to patient survival. In conclusion, MIBI offers high-parameter capability, at a sensitivity and resolution uniquely suited to understanding the complex tumor immune landscape.

BACKGROUND:Ilorasertib (ABT-348) inhibits Aurora and VEGF receptor (VEGFR) kinases. Patients with advanced solid tumours participated in a phase 1 dose-escalation trial to profile the safety, tolerability, and pharmacokinetics of ilorasertib. METHODS:Ilorasertib monotherapy was administered at 10-180 mg orally once daily (Arm I, n = 23), 40-340 mg orally twice daily (Arm II, n = 28), or 8-32 mg intravenously once daily (Arm III, n = 7), on days 1, 8, and 15 of each 28-day cycle. RESULTS:Dose-limiting toxicities were predominantly related to VEGFR inhibition. The most frequent treatment-emergent adverse events ( > 30%) were: fatigue (48%), anorexia (34%), and hypertension (34%). Pharmacodynamic markers suggested that ilorasertib engaged VEGFR2 and Aurora B kinase, with the VEGFR2 effects reached at lower doses and exposures than Aurora inhibition effects. In Arm II, one basal cell carcinoma patient (40 mg twice daily (BID)) and one patient with adenocarcinoma of unknown primary site (230 mg BID) had partial responses. CONCLUSIONS:In patients with advanced solid tumours, ilorasertib treatment resulted in evidence of engagement of the intended targets and antitumour activity, but with maximum inhibition of VEGFR family kinases occurring at lower exposures than typically required for inhibition of Aurora B in tissue. CLINICAL TRIAL REGISTRATION:NCT01110486.

Progress in understanding tumor stromal biology has been constrained in part because cancer-associated fibroblasts (CAF) are a heterogeneous population with limited cell-type–specific protein markers. Using RNA expression profiling, we identified the membrane protein leucine-rich repeat containing 15 (LRRC15) as highly expressed in multiple solid tumor indications with limited normal tissue expression. LRRC15 was expressed on stromal fibroblasts in many solid tumors (e.g., breast, head and neck, lung, pancreatic) as well as directly on a subset of cancer cells of mesenchymal origin (e.g., sarcoma, melanoma, glioblastoma). LRRC15 expression was induced by TGFβ on activated fibroblasts (αSMA+) and on mesenchymal stem cells. These collective findings suggested LRRC15 as a novel CAF and mesenchymal marker with utility as a therapeutic target for the treatment of cancers with LRRC15-positive stromal desmoplasia or cancers of mesenchymal origin. ABBV-085 is a monomethyl auristatin E (MMAE)-containing antibody–drug conjugate (ADC) directed against LRRC15, and it demonstrated robust preclinical efficacy against LRRC15 stromal-positive/cancer-negative, and LRRC15 cancer-positive models as a monotherapy, or in combination with standard-of-care therapies. ABBV-085′s unique mechanism of action relied upon the cell-permeable properties of MMAE to preferentially kill cancer cells over LRRC15-positive CAF while also increasing immune infiltrate (e.g., F4/80+ macrophages) in the tumor microenvironment. In summary, these findings validate LRRC15 as a novel therapeutic target in multiple solid tumor indications and support the ongoing clinical development of the LRRC15-targeted ADC ABBV-085. Significance: These findings identify LRRC15 as a new marker of cancer-associated fibroblasts and cancers of mesenchymal origin and provide preclinical evidence for the efficacy of an antibody-drug conjugate targeting the tumor stroma. Cancer Res; 78(14); 4059–72. ©2018 AACR.

Purpose: PARP inhibitors are being developed as therapeutic agents for cancer. More than six compounds have entered clinical trials. The majority of these compounds are β-nicotinamide adenine dinucleotide (NAD+)-competitive inhibitors. One exception is iniparib, which has been proposed to be a noncompetitive PARP inhibitor. In this study, we compare the biologic activities of two different structural classes of NAD+-competitive compounds with iniparib and its C-nitroso metabolite. Experimental Design: Two chemical series of NAD+-competitive PARP inhibitors, iniparib and its C-nitroso metabolite, were analyzed in enzymatic and cellular assays. Viability assays were carried out in MDA-MB-436 (BRCA1-deficient) and DLD1−/− (BRCA2-deficient) cells together with BRCA-proficient MDA-MB-231 and DLD1+/+ cells. Capan-1 and B16F10 xenograft models were used to compare iniparib and veliparib in vivo. Mass spectrometry and the 3H-labeling method were used to monitor the covalent modification of proteins. Results: All NAD+-competitive inhibitors show robust activity in a PARP cellular assay, strongly potentiate the activity of temozolomide, and elicit robust cell killing in BRCA-deficient tumor cells in vitro and in vivo. Cell killing was associated with an induction of DNA damage. In contrast, neither iniparib nor its C-nitroso metabolite inhibited PARP enzymatic or cellular activity, potentiated temozolomide, or showed activity in a BRCA-deficient setting. We find that the nitroso metabolite of iniparib forms adducts with many cysteine-containing proteins. Furthermore, both iniparib and its nitroso metabolite form protein adducts nonspecifically in tumor cells. Conclusions: Iniparib nonselectively modifies cysteine-containing proteins in tumor cells, and the primary mechanism of action for iniparib is likely not via inhibition of PARP activity. Clin Cancer Res; 18(2); 510–23. ©2011 AACR.

Many established cancer therapies involve DNA-damaging chemotherapy or radiotherapy. The DNA repair capacity of the tumor represents a common mechanism used by cancer cells to survive DNA-damaging therapy. Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that is activated by DNA damage and has critical roles in DNA repair. Inhibition of PARP potentiates the activity of DNA-damaging agents such as temozolomide, topoisomerase inhibitors and radiation in both in vitro and in vivo preclinical models. Recently, several PARP inhibitors have entered clinical trials either as single agents or in combination with DNA-damaging chemotherapy. Because PARP inhibitors are not cytotoxic, a biomarker assay is useful to guide the selection of an optimal biological dose. We set out to develop an assay that enables us to detect 50% PAR reduction in human tumors with 80% power in a single-plate assay while assuring no more than a 10% false-positive rate. We have developed and optimized an enzyme-linked immunosorbent assay (ELISA) to measure PARP activity that meets the above-mentioned criterion. This robust assay is able to detect PAR levels of 30-2000 pg/ml in both tumor and peripheral blood monocyte samples. In a B16F10 mouse syngeneic tumor model, PARP inhibitor ABT-888 potentiates the effect of temozolomide in suppressing tumor growth, and PARP activity is greatly reduced by ABT-888 at efficacious doses. In summary, the ELISA assay described here is suitable for biomarker studies in clinical trials of PARP inhibitors.

Preclinical research has provided a strong rationale for employing PARP inhibitors (PARPi) as chemosensitisers in combination with cytotoxic agents, radiosensitisers in combination with radiation, as well as monotherapy to induce synthetic lethality in human malignancies. The primary aim of this chapter is to describe the rationale and preclinical pharmacology of potent PARPi (such as veliparib, rucaparib and olaparib) as chemosensitisers studied in cytotoxic combination regimens. Additional aims are to review: (1) emerging strategies for employing PARPi as monotherapy to induce synthetic lethality; and (2) potentially novel mechanisms by which PARPi may enhance antitumour therapy.

Intracerebral infection of susceptible mice with Theiler's murine encephalomyelitis virus (TMEV) induces immune-mediated demyelinating disease and this system serves as a relevant infectious model for human multiple sclerosis. It was previously shown that beta2M-deficient C57BL/6 mice lacking functional CD8+ T cells display increased viral persistence and enhanced susceptibility to TMEV-induced demyelination, and yet the majority of mice are free of clinical signs. To understand the mechanisms involved in this general resistance of C57BL/6 mice in the absence of CTL responses, mice (muMT) deficient in the B-cell compartment lacking membrane IgM molecules were treated with anti-CD8 antibody and then infected with TMEV. Although little difference in the proliferative responses of peripheral T cells to UV-inactivated TMEV and the resistance to demyelinating disease was observed between virus-infected muMT and control B6 mice, the levels of CD4(+) T cells were higher in the CNS of muMT mice. However, after treatment with anti-CD8 antibody, 100% of the mice displayed clinical gray matter disease and prolonged viral persistence in muMT mice, while only 10% of B6 mice showed clinical symptoms and very low viral persistence. Transfusion of sera from TMEV-infected B6 mice into anti-CD8 antibody-treated muMT mice partially restored resistance to virus-induced encephalitis. These results indicate that the early anti-viral antibody response is also important in the protection from TMEV-induced encephalitis particularly in the absence of CD8+ T cells.

Purpose: To evaluate the preclinical pharmacokinetics, antitumor efficacy, and mechanism of action of a novel orally active farnesyltransferase inhibitor, ABT-100. Experimental Design: In vitro sensitivity of a panel of human cell lines was determined using proliferation and clonogenic assays. In vivo efficacy of ABT-100 was evaluated in xenograft models (flank or orthotopic) by assessing angiogenesis, proliferation, and apoptosis in correlation with pharmacokinetics. Efficacy of the racemate of ABT-100 (A-367074) was also compared with R115777 (tipifarnib). Results: ABT-100 inhibited proliferation of cells in vitro carrying oncogenic H-Ras (EJ-1 bladder; IC50 2.2 nmol/L), Ki-Ras (DLD-1 colon, MDA-MB-231 breast, HCT-116 colon, and MiaPaCa-2 pancreatic; IC50 range, 3.8-9.2 nmol/L), and wild-type Ras (PC-3 and DU-145; IC50, 70 and 818 nmol/L, respectively) as well as clonogenic potential. ABT-100 shows 70% to 80% oral bioavailability in mice. ABT-100 regressed EJ-1 tumors (2-12.5 mg/kg/d s.c., every day for 21 days) and showed significant efficacy in DLD-1, LX-1, MiaPaCa-2, or PC-3 tumor-bearing mice (6.25-50 mg/kg/d s.c. once daily or twice daily orally). A-367074 showed equivalent efficacy to R115777 given at approximately one-fourth the total dose of R115777 for a shorter duration (EJ-1 and LX-1). Antitumor activity was associated with decreased cell proliferation (Ki-67), increased apoptosis (terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling), and decreased angiogenesis. A reduction in tumor angiogenic cytokine levels (vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8) correlated with a reduction in tumor vascularity (CD31). Conclusions: Overall, ABT-100 has an acceptable pharmacokinetic profile, is well tolerated, and possesses broad-spectrum antitumor activity against a series of xenograft models similar to farnesyltransferase inhibitors in clinical development; therefore, it is an attractive candidate for clinical evaluation.

We have investigated the potential effects of H-2 and T-cell receptor (TCR) V beta family genes on induction of T-cell immunity and susceptibility to virally induced demyelinating disease by using BALB.S (H-2K(s)A(s)D(s)) and BALB.S 3 R (H-2K(s)A(s)D(d)/L(d)) mice. These parameters were compared with those of highly susceptible SJL/J (H-2K(s)A(s)D(s)) mice that contain only one-half of TCR V beta family genes compared with the above-mentioned strains. Our results demonstrate that BALB.S but not BALB.S 3 R mice are susceptible similar to SJL/J mice. Although the level of CD4(+) T-cell infiltration to the CNS was elevated in susceptible mice, virus-specific immune responses restricted with H-2(s) were similar in these mice. No preferential use of V beta families associated with differences in the major histocompatibility complex (MHC) components was apparent. However, the pattern and sequence of CDR 3 distribution shows T-cell clonal accumulation in the CNS associated with the H-2 components. Further anti-CD8 antibody treatment of resistant BALB.S 3 R mice abrogated resistance to demyelinating disease, indicating that CD8(+) T cells restricted with H-2D(d)/L(d) are most likely to exert resistance in BALB.S 3 R mice. These studies indicated that TCR V beta and MHC class II genes are the secondary to a particular MHC class I gene expression in susceptibility to virally induced demyelinating disease.

Although the causative agents of human multiple sclerosis (MS) are not known, it is suspected that a viral infection may be associated with the initiation of the disease. Several viral disease models in mice have been studied to understand the pathogenesis of demeylination. In particular, Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) has been extensively studied as a relevant model. Various cytokines and chemokines are produced upon viral infection by different cell types, including antigen-presenting cells (APCs) such as macrophages; dendritic cells (DCs); and glial cells, such as astrocytes, microglia, and oligoden-drocytes. The upregulation of the corresponding molecules are also found in MS and are likely to play an important role in the protection and/or pathogenesis of chronic inflammatory demyelinating disease. In this review, the type of cells and molecules, gene-activation mechanisms as well as their potential roles in protection and pathogenesis will be discussed.

Infection with different picornaviruses can cause meningitis/encephalitis in humans and experimental animals. To investigate the mechanisms of such inflammatory diseases, potential chemokine gene activation in human astrocytes was investigated following infection with Theiler's murine encephalomyelitis virus (TMEV), coxsackievirus B3 (CVB3), or coxsackievirus B4 (CVB4). We report that all these viruses are potent inducers for the expression of interleukin‐8 (IL‐8) and monocyte chemoattractant protein‐1 (MCP‐1) genes in primary human astrocytes, as well as in an established astrocyte cell line (U‐373MG). Further studies indicated that both activator protein‐1 (AP‐1) and NF‐κB transcription factors are required in the activation of chemokine genes in human astrocytes infected with various picornaviruses. Interestingly, the pattern of activated chemokine genes in human astrocytes is quite restricted compared to that in mouse astrocytes infected with the same viruses, suggesting species differences in gene activation. This may result in potential differences in the pathogenic outcome in each species. © 2003 Wiley‐Liss, Inc.

Theiler's virus induces an immune-mediated demyelinating disease similar to human multiple sclerosis. Studies have shown the importance of genetic factors like major histocompatibility complex (MHC), T-cell receptor, and gender in the susceptibility to the disease (21). Intracerebral infection of susceptible strains such as SJL/J mice results in an acute, polio-like phase of the gray matter that later develops into a chronic demyelination in the white matter manifested by severe hind limb paralysis and incontinence (25). Demyelination is characterized by mononuclear cell infiltration with the primary involvement of activated macrophages that correlates with virus-specific delayed-type hypersensitivity responses (7, 51). The Theiler's murine encephalomyelitis virus (TMEV)-specific T-cell responses to viral determinants are well characterized in this Th1-mediated disease (21). However, the early innate immune response of central nervous system (CNS)-resident cells that lead to the proinflammatory milieu critical in the recruitment and development of virus-specific Th1 responses, are largely unknown. An innate immune response to viral and bacterial infection often results in the production of immune molecules, including cytokines, chemokines, MHC, and enzymes, etc., that act in concert to control the infectious agents (13). These very same molecules can however induce dysregulated inflammation that leads to target tissue destruction (9, 26). Recent studies suggest that toll-like receptors are involved in the induction of innate immune responses following exposure to bacterial and/or viral components (18, 34). In particular, TLR-3 (as well as double-stranded RNA [dsRNA]-dependent protein kinase [PKR]) is involved in activation of a variety of proinflammatory cellular genes through the NF-κB pathway upon recognition of dsRNA, a replication intermediate of TMEV (2, 55). NF-κB represents a family of dimeric transcription factors that play a central role in these inflammatory responses by regulation of gene expression and inhibition of apoptosis (11, 20). The sequestration of NF-κB by IκB in the cytoplasm and IκB phosphorylation leading to proteasomal degradation resulting in activation and translocation of NF-κB to the nucleus is essential in the transcription of many proinflammatory chemokines and cytokines such as IP-10, RANTES, monocyte chemotactic protein 1 (MCP-1), interleukin-10 (IL-10), tumor necrosis factor alpha (TNF-α), and IL-6, (3, 4). Many investigators have extensively investigated the potential roles of cytokines in TMEV-induced, immune-mediated inflammatory demyelination (5, 6, 21, 44, 49). The accumulation of proinflammatory cytokines has been demonstrated in the CNS of TMEV-infected mice and precedes that of anti-inflammatory cytokines (IL-4 and IL-10) during the course of disease (37). Recent studies have also shown sustained upregulation of proinflammatory cytokines (gamma interferon [IFN-γ], IL-6, IL-12, and TNF-α) and transforming growth factor β, downregulating cytolytic responses in the CNS of SJL mice compared to resistant C57BL/6 mice. These results correlate well with susceptibility to demyelinating disease (6). However, the mechanisms that initiate and expand cytokine gene expression in the CNS inflammatory disease induced following TMEV infection are less clear. We and others have previously reported that TMEV is able to directly activate selective chemokine and cytokine genes in the CNS-resident glial cell populations (32, 36, 38, 46). However, the range of cytokines induced and the signal transduction mechanisms involved in this virus-mediated cytokine gene activation has not been previously explored. In order to delineate the potential pathogenic role of the innate immune response in virus-induced CNS demyelination, we have examined the molecular mechanisms underlying early cytokine gene activation after TMEV infection of primary astrocyte cultures. Here we demonstrate the ability of TMEV to directly induce selective proinflammatory cytokines (IL-12, IL-1, IL-6, TNF-α, and IFN-β) in glial cells. The expression of cytokine genes was evident for some, but not for all, as early as 30 min postinfection, and the activation of NF-κB was demonstrated within 5 min of TMEV infection. Additionally, various NF-κB inhibitors, as well as an IκB super-repressor, were shown to inhibit TMEV-induced cytokine gene expression. These results clearly indicate that NF-κB activation is necessary in TMEV-induced cytokine gene activation. The PKR as well as IFN-α/β pathways do not appear to play a major role in the cytokine gene activation. Altogether, these results strongly support the crucial role of CNS resident cells in the initiation of inflammatory demyelination by production of various proinflammatory cytokines via an NF-κB-dependent innate immune response to TMEV infection.

Despite clinical efficacy, current approved agents targeting EGFR are associated with on-target toxicities as a consequence of disrupting normal EGFR function. MAb 806 is a novel EGFR antibody that selectively targets a tumor-selective epitope suggesting that a mAb 806-based therapeutic would retain antitumor activity without the on-target toxicities associated with EGFR inhibition. To enable clinical development, a humanized variant of mAb 806 designated ABT-806 was generated and is currently in phase 1 trials. We describe the characterization of binding and functional properties of ABT-806 compared with the clinically validated anti-EGFR antibody cetuximab. ABT-806 binds the mutant EGFRvIII with high affinity and, relative to cetuximab, exhibits increased potency against glioblastoma multiforme cell line and patient-derived xenografts expressing this form of the receptor. ABT-806 also inhibits the growth of squamous cell carcinoma xenograft models expressing high levels of wild-type EGFR, associated with inhibition of EGFR signaling, although higher doses of ABT-806 than cetuximab are required for similar activity. ABT-806 enhances in vivo potency of standard-of-care therapies used to treat glioblastoma multiforme and head and neck squamous cell carcinoma. An indium-labeled version of ABT-806, [111In]-ABT-806, used to investigate the relationship between dose and receptor occupancy, revealed greater receptor occupancy at lowers doses in an EGFRvIII-expressing model and significant uptake in an orthotopic model. Collectively, these results suggest that ABT-806 may have antitumor activity superior to cetuximab in EGFRvIII-expressing tumors, and similar activity to cetuximab in tumors highly overexpressing wild-type EGFR with reduced toxicity. Mol Cancer Ther; 14(5); 1141–51. ©2015 AACR.

To elucidate the early events in Theiler's virus-induced demyelination, a model for human multiple sclerosis (MS), chemokine gene activation in the central nervous system (CNS) resident cells upon viral infection was investigated. Viral infection selectively upregulated RANTES and IP-10 gene expression in primary astrocyte cultures and broader chemokine genes in oligodendrocyte and microglia cultures. Both RANTES and IP-10 were stimulated by proinflammatory cytokine interferon-gamma (IFNgamma), but only RANTES by tumor necrosis factor alpha (TNFalpha), suggesting that virus infection induces chemokines overlapping with those inducible by proinflammatory cytokines. These results suggest that glial cells, astrocytes in particular, may be critical for early recruitment of inflammatory cells in the initiation of virus-induced, immune-mediated demyelination.

Theiler's murine encephalomyelitis virus (TMEV) infection of the central nervous system (CNS) induces a chronic, progressive demyelinating disease in susceptible mouse strains characterized by inflammatory mononuclear infiltrates and spastic hind limb paralysis. Our lab has previously demonstrated a critical role for TMEV- and myelin-specific CD4(+) T cells in initiating and perpetuating this pathology. It has however, also been shown that the MHC class I loci are associated with susceptibility/resistance to TMEV infection and persistence. For this reason, we investigated the contribution of CD8(+) T cells to the TMEV-induced demyelinating pathology in the highly susceptible SJL/J mouse strain. Here we show that beta2M-deficient SJL mice have similar disease incidence rates to wild-type controls, however beta2M-deficient mice demonstrated earlier onset of clinical disease, elevated in vitro responses to TMEV and myelin proteolipid (PLP) epitopes, and significantly higher levels of CNS demyelination and macrophage infiltration at 50 days post-infection. beta2M-deficient mice also displayed a significant elevation in persisting viral titers, as well as an increase in macrophage-derived pro-inflammatory cytokine mRNA expression in the spinal cord at this same time point. Taken together, these results indicate that CD8(+) T cells are not required for clinical or histologic disease initiation or progression in TMEV-infected SJL mice. Rather, these data stress the critical role of CD4(+) T cells in this capacity and further emphasize the potential for CD8(+) T cells to contribute to protection from TMEV-induced demyelination.

Theiler's murine encephalomyelitis virus-induced demyelinating disease has been extensively studied as an attractive infectious model for human multiple sclerosis. Virus-specific inflammatory Th1 cell responses followed by autoimmune responses to myelin antigens play a crucial role in the pathogenic processes leading to demyelination. Antibody and cytotoxic T cells (CTL) responses to virus appears to be primarily protective from demyelinating disease. Although the role of Th1 and CTL responses in the induction of demyelinating disease is controversial, assessment of cytokines produced locally in the central nervous system (CNS) during the course of disease and the effects of altered inflammatory cytokine levels strongly support the importance of Th1 responses in this virus-induced demyelinating disease. Induction of various chemokines and cytokines in different glial and antigen presenting cells upon viral infection appears to be an important initiation mechanism for inflammatory Th1 responses in the CNS. Coupled with the initial inflammatory responses, viral persistence in the CNS may be a critical factor for sustaining inflammatory responses and consequent immune-mediated demyelinating disease.

Intracerebral inoculation of Theiler’s murine encephalomyelitis virus (TMEV) into susceptible mouse strains results in a chronic, immune-mediated demyelinating disease similar to human multiple sclerosis. Here, we examined the role of astrocytes as an APC population in TMEV-induced demyelination and assessed the potential consequences of T cell activation following Ag presentation. IFN-γ-pretreated astrocytes were able to process and present all the predominant T cell epitopes of TMEV to virus-specific T cell hybridomas, clones, as well as bulk T cells. Despite low levels of proliferation of T cells due to prostaglandins produced by astrocytes, such Ag presentation by activated astrocytes induced the production of IFN-γ, a representative proinflammatory cytokine, in TMEV-specific Th cell clones derived from the CNS of virus-infected mice. Furthermore, these Th cell clones mediate lysis of the astrocytes in vitro in a Fas-dependent mechanism. TUNEL staining of CNS tissue demonstrates the presence of apoptotic GFAP+ cells in the white matter of TMEV-infected mice. These results strongly suggest that astrocytes could play an important role in the pathogenesis of TMEV-induced demyelination by activating T cells, subsequently leading to T cell-mediated apoptosis of astrocytes and thereby compromising the blood-brain barrier.

Intracerebral inoculation of susceptible strains of mice with Theiler’s murine encephalomyelitis virus (TMEV) results in immune-mediated demyelination. Three major T-cell epitopes have previously been identified within the VP1 (VP1 233–250 ), VP2 (VP2 74–86 ), and VP3 (VP3 24–37 ) capsid proteins in virus-infected SJL/J mice. These epitopes appear to account for the majority (∼90%) of major histocompatibility complex class II-restricted T-cell responses to TMEV. Interestingly, the effect of immunization with synthetic peptides bearing the predominant T-cell epitopes on the course of TMEV-induced demyelination indicates that T cells reactive to the VP1 and VP2 epitopes, but not VP3, accelerate the pathogenesis of demyelination. The predominant pathogenic role of the T cells is verified by similar immunization with the fusion proteins containing the entire individual capsid proteins. The order of appearance and level of T cells specific for the individual epitopes during the course of demyelination are similar to each other. However, cytokine profiles of T cells from virus-infected mice indicate that T cells specific for the VP1 (and perhaps the VP2) epitope are Th1, whereas T cells reactive to VP3 are primarily Th2. These results suggest that Th1-type cells specific for VP1 and VP2 are involved in the pathogenesis of viral demyelination induced by TMEV. Thus, a predominance of Th1-inducing viral epitopes is likely critical for the pathogenesis of demyelination.

Intracerebral infection of susceptible mouse strains with Theiler's murine encephalomyelitis virus (TMEV) results in an immune-mediated demyelinating disease (TMEV-IDD) similar to human multiple sclerosis (MS). Although the etiology of MS remains unknown, a role of an infectious agent has been implicated in its onset. Previously we have shown the ability of bacterial lipopolysaccharide (LPS) to alter susceptibility to TMEV-IDD in genetically resistant C57BL/6 mice. In this study, the potential of LPS to alter pathogenicity of a low/non-pathogenic variant of TMEV was investigated. After intraperitoneal treatment of genetically susceptible SJL/J mice with LPS before and during viral infection, 80-100% of the mice developed clinical symptoms, while without LPS treatment none of the mice were affected. However, clinical severity in these LPS-treated mice was much milder than the level induced by the wild type pathogenic virus. Increased susceptibility to the disease after LPS treatment did not correlate with splenic T cell proliferative responses against viral antigens. However, by reverse transcriptase polymerase chain reaction (RT-PCR) analyses, an early increase in the production of Th1-type proinflammatory cytokine messages (e.g., interferon-gamma [IFN-gamma] and enhancement of viral persistence was observed in the CNS of LPS-treated, virus-infected animals as compared to mice infected with the variant virus alone. These results indicate that environmental factors such as a bacterial infection (e.g., LPS) promoting proinflammatory cytokine production can significantly enhance the pathogenicity of demyelination induced by a normally non-pathogenic virus.

We examined the development of the aquatic N2-fixing symbiosis between Rhizobium sp. (itNeptunia) and roots of Neptunia natans L. f. (Druce) (previously N. oleracea Lour.) under natural and laboratory conditions. When grown in its native marsh habitat, this unusual aquatic legume does not develop root hairs, the primary sites of rhizobial infection for most temperate legumes. Under natural conditions, the aquatic plant floats and develops nitrogen-fixing nodules at emergence of lateral roots on the primary root and on adventitious roots at stem nodes, but not from the stem itself. Cytological studies using various microscopies revealed that the mode of root infection involved an intercellular route of entry followed by an intracellular route of dissemination within nodule cells. After colonizing the root surface, the bacteria entered the primary root cortex through natural wounds caused by splitting of the epidermis and emergence of young lateral roots, and then stimulated early development of nodules at the base of such roots. The bacteria entered the nodule through pockets between separated host cells, then spread deeper in the nodule through a narrower intercellular route, and eventually evoked the formation of infection threads that penetrated host cells and spread throughout the nodule tissue. Bacteria were released from infection droplets at unwalled ends of infection threads, became enveloped by peribacteroid membranes, and transformed into enlarged bacteroids within symbiosomes. In older nodules, the bacteria within symbiosomes were embedded in an unusual, extensive fibrillar matrix. Cross-inoculation tests of 18 isolates of rhizobia from nodules of N. natans revealed a host specificity enabling effective nodulation of this aquatic legume, with lesser affinity for Medicago sativa and Ornithopus sp., and an inability to nodulate several other crop legume species. Acetylene reduction (N2 fixation) activity was detected in nodules of N. natans growing in aquatic habitats under natural conditions in Southern India. These studies indicate that a specific group of Rhizobium sp. (Neptunia) occupies a unique ecological niche in aquatic environments by entering into a N2-fixing root-nodule symbiosis with Neptunia natans.

In an effort to identify kinase inhibitors with dual KDR/Aurora B activity and improved aqueous solubility compared to the Abbott dual inhibitor ABT-348, a series of novel pyrazole pyrimidines structurally related to kinase inhibitor AS703569 were prepared. SAR work provided analogs with significant cellular activity, measureable aqueous solubility and moderate antitumor activity in a mouse tumor model after weekly ip dosing. Unfortunately these compounds were pan-kinase inhibitors that suffered from narrow therapeutic indices which prohibited their use as antitumor agents.

The anticancer drugs cisplatin and carboplatin have been shown to activate murine peritoneal macrophages in vivo and in vitro. These activated macrophages have enhanced tumoricidal activity mediated by extension and contact formation with the tumor cells leading to an increase and transfer of lysosomes with eventual lysis of the tumor cells. Cisplatin (10 micrograms/ml) or carboplatin (50 micrograms/ml) for 2 and 24 h treatment of macrophages in vitro, in addition, show a significant increase in the release of various cytolytic factors, like hydrogen peroxide, superoxide anion, interleukin-1 alpha, lysozyme and beta-N-hexoseaminidase, that are also responsible for the destruction of tumor cells.

Longitudinal changes of 3'-[(18) F]fluoro-3'-deoxythymidine (FLT) and 2-deoxy-2-[(18) F]fluoro-D-glucose (FDG) in response to irinotecan therapy in an animal model of colorectal cancer were compared.SCID/CB-17 mice with HCT116 tumors were treated with 50 mg/kg irinotecan by intraperitoneal injection weekly for 3 weeks. FLT and FDG-positron emission tomography (PET) were performed at baseline, the day after each treatment, and 5 days after the first treatment. Proliferation and apoptosis were evaluated by immunohistochemistry (IHC) after day 15 of imaging.Irinotecan treatment resulted in a suppression of tumor growth. Tumor FLT uptake was decreased the day after each treatment but to a lesser extent 5 days after the first treatment. FDG uptake increased the day after each treatment with a continuous increase throughout the experiment. IHC analysis of phospho-H3 and Ki67 confirmed FLT-PET results, indicating a decrease in proliferation the day after the final irinotecan treatment. Increased apoptosis monitored by caspase-3 was observed after day 15 with irinotecan treatment.FLT-PET may be a better method than FDG-PET for assessing treatment response to irinotecan. Changes in imaging occur before changes in tumor volume.

Aurora kinase B inhibitors induce apoptosis secondary to polyploidization and have entered clinical trials as an emerging class of neocytotoxic chemotherapeutics. We demonstrate here that polyploidization neutralizes Mcl-1 function, rendering cancer cells exquisitely dependent on Bcl-XL/-2. This “addiction” can be exploited therapeutically by combining aurora kinase inhibitors and the orally bioavailable BH3 mimetic, ABT-263, which inhibits Bcl-XL, Bcl-2, and Bcl-w. The combination of ABT-263 with aurora B inhibitors produces a synergistic loss of viability in a range of cell lines of divergent tumor origin and exhibits more sustained tumor growth inhibition in vivo compared with aurora B inhibitor monotherapy. These data demonstrate that Bcl-XL/-2 is necessary to support viability during polyploidization in a variety of tumor models and represents a druggable molecular vulnerability with potential therapeutic utility.

Purpose: ABT-888, currently in phase 2 trials, is a potent oral poly(ADP-ribose) polymerase inhibitor that enhances the activity of multiple DNA-damaging agents, including temozolomide (TMZ). We investigated ABT-888+TMZ combination therapy in multiple xenograft models representing various human tumors having different responses to TMZ. Experimental Design: ABT-888+TMZ efficacy in xenograft tumors implanted in subcutaneous, orthotopic, and metastatic sites was assessed by tumor burden, expression of poly(ADP-ribose) polymer, and O6-methylguanine methyltransferase (MGMT). Results: Varying levels of ABT-888+TMZ sensitivity were evident across a broad histologic spectrum of models (55-100% tumor growth inhibition) in B-cell lymphoma, small cell lung carcinoma, non–small cell lung carcinoma, pancreatic, ovarian, breast, and prostate xenografts, including numerous regressions. Combination efficacy in otherwise TMZ nonresponsive tumors suggests that TMZ resistance may be overcome by poly(ADP-ribose) polymerase inhibition. Profound ABT-888+TMZ efficacy was seen in experimental metastases models that acquired resistance to TMZ. Moreover, TMZ resistance was overcome in crossover treatments, indicating that combination therapy may overcome acquired TMZ resistance. Neither tumor MGMT, mismatch repair, nor poly(ADP-ribose) polymer correlated with the degree of sensitivity to ABT-888+TMZ. Conclusions: Robust ABT-888+TMZ efficacy is observed across a spectrum of tumor types, including orthotopic and metastatic implantation. As many TMZ nonresponsive tumors proved sensitive to ABT-888+TMZ, this novel combination may broaden the clinical use of TMZ beyond melanoma and glioma. Although TMZ resistance may be influenced by MGMT, neither MGMT nor other mechanisms of TMZ resistance (mismatch repair) precluded sensitivity to ABT-888+TMZ. Underlying mechanisms of TMZ resistance in these models are not completely understood but likely involve mechanisms independent of MGMT.(Clin Cancer Res 2009;15(23):7277–90)

ABT-888 is a potent, orally bioavailable PARP-1/2 inhibitor shown to potentiate DNA damaging agents. The ability to potentiate temozolomide (TMZ) and develop a biological marker for PARP inhibition was evaluated in vivo. Doses/schedules that achieve TMZ potentiation in the B16F10 syngeneic melanoma model were utilized to develop an ELISA to detect a pharmacodynamic marker, ADP ribose polymers (pADPr), after ABT 888 treatment. ABT-888 enhanced TMZ antitumor activity, in a dose-proportional manner with no observed toxicity (44-75% tumor growth inhibition vs. TMZ monotherapy), but did not show single agent activity. Extended ABT-888 dosing schedules showed no advantage compared to simultaneous TMZ administration. Efficacy correlated with plasma/tumor drug concentrations. Intratumor drug levels correlated with a dose-proportional/time-dependent reduction in pADPr. Potentiation of TMZ activity by ABT-888 correlated with drug levels and inhibition of PARP activity in vivo. ABT-888 is in Phase 1 trials using a validated ELISA based on the assay developed here to assess pharmacological effect.

Poly(ADP-ribose) polymerase (PARP) senses DNA breaks and facilitates DNA repair via the polyADP-ribosylation of various DNA binding and repair proteins. We explored the mechanism of potentiation of temozolomide cytotoxicity by the PARP inhibitor ABT-888. We showed that cells treated with temozolomide need to be exposed to ABT-888 for at least 17 to 24 hours to achieve maximal cytotoxicity. The extent of cytotoxicity correlates with the level of double-stranded DNA breaks as indicated by γH2AX levels. In synchronized cells, damaging DNA with temozolomide in the presence of ABT-888 during the S phase generated high levels of double-stranded breaks, presumably because the single-stranded DNA breaks resulting from the cleavage of the methylated nucleotides were converted into double-stranded breaks through DNA replication. As a result, treatment of temozolomide and ABT-888 during the S phase leads to higher levels of cytotoxicity. ABT-888 inhibits poly(ADP-ribose) formation in vivo and enhances tumor growth inhibition by temozolomide in multiple models. ABT-888 is well tolerated in animal models. ABT-888 is currently in clinical trials in combination with temozolomide. (Mol Cancer Res 2008;6(10):1621–9)

Purpose: To evaluate the preclinical pharmacokinetics and antitumor efficacy of a novel orally bioavailable poly(ADP-ribose) polymerase (PARP) inhibitor, ABT-888. Experimental Design: In vitro potency was determined in a PARP-1 and PARP-2 enzyme assay. In vivo efficacy was evaluated in syngeneic and xenograft models in combination with temozolomide, platinums, cyclophosphamide, and ionizing radiation. Results: ABT-888 is a potent inhibitor of both PARP-1 and PARP-2 with Kis of 5.2 and 2.9 nmol/L, respectively. The compound has good oral bioavailability and crosses the blood-brain barrier. ABT-888 strongly potentiated temozolomide in the B16F10 s.c. murine melanoma model. PARP inhibition dramatically increased the efficacy of temozolomide at ABT-888 doses as low as 3.1 mg/kg/d and a maximal efficacy achieved at 25 mg/kg/d. In the 9L orthotopic rat glioma model, temozolomide alone exhibited minimal efficacy, whereas ABT-888, when combined with temozolomide, significantly slowed tumor progression. In the MX-1 breast xenograft model (BRCA1 deletion and BRCA2 mutation), ABT-888 potentiated cisplatin, carboplatin, and cyclophosphamide, causing regression of established tumors, whereas with comparable doses of cytotoxic agents alone, only modest tumor inhibition was exhibited. Finally, ABT-888 potentiated radiation (2 Gy/d × 10) in an HCT-116 colon carcinoma model. In each model, ABT-888 did not display single-agent activity. Conclusions: ABT-888 is a potent inhibitor of PARP, has good oral bioavailability, can cross the blood-brain barrier, and potentiates temozolomide, platinums, cyclophosphamide, and radiation in syngeneic and xenograft tumor models. This broad spectrum of chemopotentiation and radiopotentiation makes this compound an attractive candidate for clinical evaluation.

Multiple sclerosis involves inflammatory immune responses in the central nervous system (CNS) and is considered as an autoimmune disease potentially associated with viral infection. The majority of experimental models rely heavily on the autoimmune components since similar diseases can be induced following immunization with various myelin antigens. A very attractive alternative model is the Theiler’s murine encephalomyelitis virus-induced demyelinating disease. This disease is primarily a CD4+ T cell-mediated, inflammatory demyelinating disease induced following viral infection. Virus-specific inflammatory Th1 cell responses, rather than cytotoxic T lymphocyte response, play a critical role in the pathogenic immune responses. The major pathogenic epitopes have been identified and these are correlated with a Th1 type response to the epitopes following viral infection. In addition, the initial virus-specific immune response is followed by the autoimmune responses to myelin antigens. Assessment of cytokines produced locally in the CNS during the course of disease suggests involvement of inflammatory cytokines in the disease. Furthermore, the manipulation of inflammatory cytokine levels by administration of either recombinant cytokines or antibodies to the cytokines strongly influences the induction and/or progression of disease, supporting the importance of these inflammatory cytokines in this virus-induced demyelinating disease.

Analysis of tissue culture supernatants collected from cisplatin (10 micrograms/ml) and carboplatin (50 micrograms/ml)-treated macrophages show enhanced activity of interleukin-1 alpha and tumor necrosis factor-alpha. Cytotoxicity of these supernatants was demonstrated using mouse sarcoma-180 cells. These results demonstrate the ability of cisplatin and carboplatin to enhance the immune system suggestive of yet another mechanism of their action in the regression of tumors.

The Aurora kinases are a family of serine/threonine kinases that mediate essential functions in cell division. Aurora A depletion results in accumulation of cells in the G2/M phase and apoptosis. Inhibition of Aurora B/C results in abnormal cell division, polyploidy, resulting in apoptosis, therefore, Aurora kinases present an attractive target for chemotherapy. Cells treated with aurora inhibitors enter mitosis with normal kinetics but fail to undergo cytokinesis due to mitotic spindle checkpoint disruption. ABT-348 is a novel adenosine triphosphate (ATP)-competitive inhibitor of Aurora A, Aurora B, and Aurora C (Enzyme IC50 A=116, B=5, C= 1 nM) and a potent inhibitor of all members of the VEGF and PDGF family of receptor tyrosine kinases (RTKs). Despite significant advances in the epidemiological, genetic and biological understanding of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), the basic therapeutic approach has not substantially changed for the last 10-15 years, so most patients still die of this disease. Here we demonstrate profound in vivo efficacy (with regressions) of ABT-348 in both AML (MV-4-11, FLT3 mutant expressing the internal tandem duplication with constitutive kinase activation) and MDS (SKM-1) xenograft models. MV-4-11 tumor-bearing SCID mice were treated at 6.25, 12.5 and 25 mg/kg/day, p.o., q7d x 3 (%TGI ratios on day 30 were 80, 86 and 94%, respectively). SKM-1 tumor-bearing SCID mice were treated at 6.25, 12.5 and 25 mg/kg/day, p.o., q7d x 3 (% tumor growth inhibition or TGI ratios on day 30 were 38, 59 and 80%, respectively). In addition, ABT-348 provided additive effects when combined with cytarabine, decitabine or doxorubicin compared to cytotoxic monotherapies. The treatments were well tolerated with no animal health concerns observed indicating the feasibility of ABT-348 combination strategies in the clinic. Currently ABT-348 is being evaluated (monotherapy and in combination with cytotoxic therapies) in the HL-60 acute promyelocytic leukemia xenograft model in vivo. Dose/scheduling studies for combination therapies in the SKM-1 and HL-60 xenografts are ongoing. Pharmacokinetic/pharmacodynamic biomarker analyses in these xenograft models were evaluated using phospho-H3 (an Aurora B substrate, proliferation) and cleaved caspase-3 (apoptosis) by IHC at various timepoints post single dose (1/2 hr to 5 days). A general decrease in proliferation and increase in apoptosis consistent with the mechanism of action was observed which coincided with the potent in vivo efficacy in xenograft models. Overall, ABT-348 is a potent, oral Aurora kinase inhibitor, demonstrating robust in antitumor activity in AML and MDS xenograft models with a good safety profile that warrants investigation in the clinic. ABT-348 is currently undergoing Phase I clinical trials in advanced hematologic malignancies. 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 858. doi:1538-7445.AM2012-858

The Aurora kinases (Aurora A, B, and C) play essential roles in regulating cell division in mammalian cells and their over-expression in diverse tumor types makes them appealing oncology targets. ABT-348 is a novel, ATP-competitive, multi-targeted kinase inhibitor that exhibits potent activity in multiple solid tumor-derived and leukemia cell lines. ABT-348 is active against Aurora B (IC50 7 nM) and Aurora C (IC50 1 nM), Aurora A (IC50 120 nM). The activity against Aurora B is demonstrated by inhibition of histone H3 phosphorylation and induction of polyploidy. ABT-348 is also active against Aurora-B Y156H, a mutant resistant to other Aurora-B inhibitors. In addition, ABT-348 potently inhibits most members of the VEGFR and PDGFR family of receptor tyrosine kinases, which play a critical role in stromal angiogenesis. In contrast to other Aurora kinase inhibitors, the cellular efficacy of ABT-348 is retained in cells over-expressing P-glycoprotein (Pgp) or breast cancer resistant protein (BCRP), indicating that ABT-348 is not a substrate for these commonly upregulated ATP-binding cassette drug transporters. Consistent with these in vitro studies, ABT-348 was broadly efficacious as a single agent against a wide range of tumor types in vivo, including 3 multi-drug resistant xenograft models. In summary, the potent activity and unique kinase selectivity of ABT-348 against the Aurora kinases and VEGF and PDGF receptor tyrosine kinases, engender its ability to block multiple mechanisms of tumor progression. In addition, our data provide evidence that ABT-348 may be active in tumors resistant to other well-characterized inhibitors targeting Aurora-B. ABT-348 is presently under clinical evaluation in adult patients with advanced solid and hematological neoplasms. 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 1818. doi:1538-7445.AM2012-1818

The Aurora kinases are a family of serine/threonine kinases that mediate multiple essential functions in cell division. Aurora A depletion results in accumulation of cells in the G2/M phase and apoptosis. Inhibition of Aurora B/C results in abnormal cell division, polyploidy, followed by apoptosis, therefore, Aurora kinases present an attractive target for chemotherapy. Cells treated with aurora inhibitors enter mitosis with normal kinetics but fail to undergo cytokinesis due to a disruption of the mitotic spindle checkpoint. ABT-348 is a novel adenosine triphosphate (ATP)-competitive inhibitor of Aurora A, Aurora B, and Aurora C (Enzyme IC50 A=116, B=5, C= 1 nM) and a potent inhibitor of all members of the VEGF and PDGF family of receptor tyrosine kinases (RTK). Here we demonstrate profound in vivo efficacy (with regressions) in a broad spectrum of histological types (breast, colon, NSCLC, HNSCC, melanoma, ovarian, pancreatic, prostate, leukemia, lymphoma and renal; 50–94% TGI, tumor growth inhibition). The treatment was well tolerated with no animal health concerns observed. In addition, ABT-348 was also efficacious in tumor xenografts overexpressing P-gp and provided significant in vivo efficacy in comparison to competitor compounds in colon, NSCLC and ovarian xenograft models. Various dosing schemes to achieve optimal efficacy (IV, OMP and PO) were investigated. The anti-tumor efficacy of ABT-348 was not affected by the route of administration. Similar efficacy was achieved by IV, OMP or PO administration, once weekly in a dose-dependent manner. Pharmacokinetic/pharmacodynamic biomarker analysis in select tumor xenograft models were evaluated by phospho-H3 (an Aurora B substrate, proliferation marker) and cleaved caspase-3 (apoptosis marker) by IHC at various timepoints post single dose (6 hr to 9 days). Significant levels of ABT-348 were detected in the plasma and tumor. A general decrease in proliferation and increase in apoptosis consistent with the mechanism of action was observed and coincide with the potent in vivo efficacy in xenograft models. Overall, ABT-348 is a potent, oral Aurora kinase inhibitor, demonstrating robust in vitro and in vivo antitumor activity with a good safety profile that warrants investigation in the clinic. ABT-348, is currently undergoing Phase I clinical trials in both solid and hematologic malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B231.