Christina M. Annunziata

Combination Targeted Therapy With Sorafenib and Bevacizumab Results in Enhanced Toxicity and Antitumor Activity

PURPOSE Sorafenib inhibits Raf kinase and vascular endothelial growth factor (VEGF) receptor. Bevacizumab is a monoclonal antibody targeted against VEGF. We hypothesized that the complementary inhibition of VEGF signaling would have synergistic therapeutic effects. PATIENTS AND METHODS Patients had advanced solid tumors, Eastern Cooperative Oncology Group performance status of 0 to 1, and good end-organ function. A phase I dose-escalation trial of sorafenib and bevacizumab was initiated at below-recommended single-agent doses because of possible overlapping toxicity: sorafenib 200 mg orally twice daily and bevacizumab intravenously at 5 mg/kg (dose level [DL] 1) or 10 mg/kg (DL2) every 2 weeks. Additional patients were enrolled at the maximum-tolerated dose (MTD). RESULTS Thirty-nine patients were treated. DL1 was the MTD and administered in cohort 2 (N = 27). Dose-limiting toxicity in DL2 was grade 3 proteinuria and thrombocytopenia. Adverse events included hypertension, hand-foot syndrome, diarrhea, transaminitis, and fatigue. Partial responses (PRs) were seen in six (43%) of 13 patients with ovarian cancer (response duration range, 4 to 22+ months) and one of three patients with renal cell cancer (response duration, 14 months). PR or disease stabilization >or= 4 months (median, 6 months; range, 4 to 22+ months) was seen in 22 (59%) of 37 assessable patients. The majority (74%) required sorafenib dose reduction to 200 mg/d at a median of four cycles (range, one to 12 cycles). CONCLUSION Combination therapy with sorafenib and bevacizumab has promising clinical activity, especially in patients with ovarian cancer. The rapidity and frequency of sorafenib dose reductions indicates that sorafenib at 200 mg twice daily with bevacizumab 5 mg/kg every 2 weeks may not be tolerable long term, and alternate sorafenib dosing schedules should be explored.

Nuclear factor κB transcription factors are coexpressed and convey a poor outcome in ovarian cancer

Recent work has suggested a role for nuclear factor κB (NF‐κB) in the propagation of ovarian cancer cell lines, but the significance and mechanism of NF‐κB in ovarian cancer is unknown. The authors hypothesized that the NF‐κB pathway is over activated in aggressive ovarian cancers.

Phase I/Ib Study of Olaparib and Carboplatin in BRCA1 or BRCA2 Mutation-Associated Breast or Ovarian Cancer With Biomarker Analyses

BACKGROUND Olaparib has single-agent activity against breast/ovarian cancer (BrCa/OvCa) in germline BRCA1 or BRCA2 mutation carriers (gBRCAm). We hypothesized addition of olaparib to carboplatin can be administered safely and yield preliminary clinical activity. METHODS Eligible patients had measurable or evaluable disease, gBRCAm, and good end-organ function. A 3 + 3 dose escalation tested daily oral capsule olaparib (100 or 200mg every 12 hours; dose level1 or 2) with carboplatin area under the curve (AUC) on day 8 (AUC3 day 8), then every 21 days. For dose levels 3 to 6, patients were given olaparib days 1 to 7 at 200 and 400 mg every 12 hours, with carboplatin AUC3 to 5 on day 1 or 2 every 21 days; a maximum of eight combination cycles were permitted, after which daily maintenance of olaparib 400mg every12 hours continued until progression. Dose-limiting toxicity was defined in the first two cycles. Peripheral blood mononuclear cells were collected for polymorphism analysis and polyADP-ribose incorporation. Paired tumor biopsies (before/after cycle 1) were obtained for biomarker proteomics and apoptosis endpoints. RESULTS Forty-five women (37 OvCa/8 BrCa) were treated. Dose-limiting toxicity was not reached on the intermittent schedule. Expansion proceeded with olaparib 400mg every 12 hours on days 1 to 7/carboplatin AUC5. Grade 3/4 adverse events included neutropenia (42.2%), thrombocytopenia (20.0%), and anemia (15.6%). Responses included 1 complete response (1 BrCa; 23 months) and 21 partial responses (50.0%; 15 OvCa; 6 BrCa; median = 16 [4 to >45] in OvCa and 10 [6 to >40] months in BrCa). Proteomic analysis suggests high pretreatment pS209-eIF4E and FOXO3a correlated with duration of response (two-sided P < .001; Pearsons R (2) = 0.94). CONCLUSIONS Olaparib capsules 400mg every 12 hours on days 1 to 7/carboplatin AUC5 is safe and has activity in gBRCAm BrCa/OvCa patients. Exploratory translational studies indicate pretreatment tissue FOXO3a expression may be predictive for response to therapy, requiring prospective validation.

Poly (ADP-Ribose) Polymerase as a Novel Therapeutic Target in Cancer

Cancer chemotherapy exploits limitations in repairing DNA damage in order to kill proliferating malignant cells. Recent evidence suggests that cancers within and across tissue types have specific defects in DNA repair pathways, and that these defects may predispose for sensitivity and resistance to various classes of cytotoxic agents. Poly (ADP-ribose) polymerase (PARP) and BRCA proteins are central to the repair of DNA strand breaks and, when defective, lead to the accumulation of mutations introduced by error-prone DNA repair. Breast, ovarian, and other cancers develop in the setting of BRCA deficiency, and these cancers may be more sensitive to cytotoxic agents that induce DNA strand breaks, as well as inhibitors of PARP activity. A series of recent clinical trials has tested whether PARP inhibitors can achieve synthetic lethality in BRCA-pathway-deficient tumors. Future studies must seek to identify sporadic cancers that harbor genomic instability, rendering susceptibility to agents that induce additional and lethal DNA damage. Clin Cancer Res; 16(18); 4517–26. ©2010 AACR.

Vandetanib, Designed to Inhibit VEGFR2 and EGFR Signaling, Had No Clinical Activity as Monotherapy for Recurrent Ovarian Cancer and No Detectable Modulation of VEGFR2

Purpose: To evaluate clinical activity and target modulation of vandetanib in women with recurrent ovarian cancer. Experimental Design: A phase II trial of orally administered vandetanib 300 mg daily was designed to include analyses of target inhibition through paired biopsies and dynamic imaging. Core 18-gauge needle biopsies and dynamic contrast-enhanced magnetic resonance imaging were obtained before initiation of therapy and 6 weeks into therapy. Biopsy samples were subjected to reverse-phase protein lysate array endpoint analysis. Cytokine concentrations were measured by enzyme-linked immunosorbent assay in serially collected plasma samples. Results: Twelve patients entered the study, and accrual was terminated in the first stage because of lack of response or disease stabilization beyond 6 months. Adverse events included rash, diarrhea, and prolonged QT interval corrected for heart rate, but not hypertension. Exploratory analyses showed that epidermal growth factor receptor (EGFR) phosphorylation was reduced in the eight paired biopsy sets obtained; vascular endothelial growth factor (VEGF) receptor-2 phosphorylation was not consistently affected nor were dynamic contrast-enhanced MRI permeability and flow parameters. Serial plasma VEGF concentrations were variable and did not significantly change in the 11 patients assessed. Conclusions: Vandetanib 300 mg daily monotherapy had no significant clinical benefit in this disease setting. Proteomic analysis of paired biopsies detected both phosphorylated-EGFR and phosphorylated-VEGF receptor-2 in ovarian tumor tissue, but only phosphorylated-EGFR was measurably inhibited by vandetanib. Clin Cancer Res; 16(2); 664–72

Proteasome inhibitors increase tubulin polymerization and stabilization in tissue culture cells : A possible mechanism contributing to peripheral neuropathy and cellular toxicity following proteasome inhibition

Bortezomib (Velcade®), a proteasome inhibitor, is approved by the FDA for the treatment of multiple myeloma (MM). While effective, its use has been hampered by peripheral neurotoxicity of unexplained etiology. Since proteasome inhibitors alter protein degradation, we speculated that proteins regulating microtubule (MT) stability may be affected after treatment and examined MT polymerization in cells by comparing the distribution of tubulin between polymerized (P) and soluble (S) fractions. We observed increased MT polymerization following treatment of SY5Y and KCNR [neuroblastoma], HCN2, and 8226 [MM] cells, using five proteasome inhibitors; the baseline proportion of total α-tubulin in ‘P’ fractions ranged from ~41-68%, and increased to ~55-99% after treatment. Increased acetylated α-tubulin, a post-translational marker of stabilized MTs, was observed in the neural cell lines HCN1A and HCN2 and this was sustained up to 144 hours after the proteasome inhibitor was removed. Cell cycle analysis of three cell lines after treatment, showed ~50-75% increases in the G2M phase. Immunofluorescent localization studies of proteasome inhibitor treated cells did not reveal microtubule bundles in contrast to paclitaxel treated, suggesting MT stabilization via a mechanism other than direct drug binding. We examined the levels of microtubule associated proteins and observed a 1.4-3.7 fold increase in the microtubule associated protein MAP2, in HCN2 cells following treatment with proteasome inhibitors. These data provide a plausible explanation for the neurotoxicity observed clinically and raise the possibility that microtubule stabilization contributes to cytotoxicity.

Proteomics in Clinical Trials and Practice Present Uses and Future Promise

The study of clinical proteomics is a promising new field that has the potential to have many applications, including the identification of biomarkers and monitoring of disease, especially in the field of oncology. Expression proteomics evaluates the cellular production of proteins encoded by a particular gene and exploits the differential expression and post-translational modifications of proteins between healthy and diseased states. These biomarkers may be applied towards early diagnosis, prognosis, and prediction of response to therapy. Functional proteomics seeks to decipher protein-protein interactions and biochemical pathways involved in disease biology and targeted by newer molecular therapeutics. Advanced spectrometry technologies and new protein array formats have improved these analyses and are now being applied prospectively in clinical trials. Further advancement of proteomics technology could usher in an era of personalized molecular medicine, where diseases are diagnosed at earlier stages and where therapies are more effective because they are tailored to the protein expression of a patient’s malignancy.

Activation of NF-κB Signaling by Inhibitor of NF-κB Kinase β Increases Aggressiveness of Ovarian Cancer

The NF-kappaB family of transcription factors has been implicated in the propagation of ovarian cancer, but the significance of constitutive NF-kappaB signaling in ovarian cancer is unknown. We hypothesized that constitutive NF-kappaB signaling defines a subset of ovarian cancer susceptible to therapeutic targeting of this pathway. We investigated the biological relevance of NF-kappaB in ovarian cancer using a small-molecule inhibitor of inhibitor of NF-kappaB kinase beta (IKKbeta) and confirmed with RNA interference toward IKKbeta. We developed a gene expression signature of IKKbeta signaling in ovarian cancer using both pharmacologic and genetic manipulation of IKKbeta. The expression of IKKbeta protein itself and the nine-gene ovarian cancer-specific IKKbeta signature were related to poor outcome in independently collected sets of primary ovarian cancers (P = 0.02). IKKbeta signaling in ovarian cancer regulated the transcription of genes involved in a wide range of cellular effects known to increase the aggressive nature of the cells. We functionally validated the effect of IKKbeta signaling on proliferation, invasion, and adhesion. Downregulating IKKbeta activity, either by a small-molecule kinase inhibitor or by short hairpin RNA depletion of IKKbeta, blocked all of these cellular functions, reflecting the negative regulation of the target genes identified. The diversity of functions controlled by IKKbeta in ovarian cancer suggests that therapeutic blockade of this pathway could be efficacious if specific IKKbeta inhibitor therapy is focused to patients whose tumors express a molecular profile suggestive of dependence on IKKbeta activity.The NF-κB family of transcription factors has been implicated in the propagation of ovarian cancer, but the significance of constitutive NF-κB signaling in ovarian cancer is unknown. We hypothesized that constitutive NF-κB signaling defines a subset of ovarian cancer susceptible to therapeutic targeting of this pathway. We investigated the biological relevance of NF-κB in ovarian cancer using a small molecule inhibitor of IKKβ, and confirmed with RNA interference towards IKKβ. We developed a gene expression signature of IKKβ signaling in ovarian cancer using both pharmacologic and genetic manipulation of IKKβ. The expression of IKKβ protein itself and the 9-gene ovarian cancer-specific IKKβ signature were related to poor outcome in independently collected sets of primary ovarian cancers (p=0.02). IKKβ signaling in ovarian cancer regulated the transcription of genes involved in a wide range of cellular effects known to increase the aggressive nature of the cells. We functionally validated the effect of IKKβ signaling on proliferation, invasion and adhesion. Downregulating IKKβ activity, either by a small molecule kinase inhibitor or by shRNA depletion of IKKβ, blocked all of these cellular functions, reflecting the negative regulation of the target genes identified. The diversity of functions controlled by IKKβ in ovarian cancer suggest that therapeutic blockade of this pathway could be efficacious if specific IKKβ inhibitor therapy is focused to patients whose tumors express a molecular profile suggestive of dependence on IKKβ activity.

The sumoylation pathway is dysregulated in multiple myeloma and is associated with adverse patient outcome

Multiple myeloma (MM) is a plasma cell neoplasm that proceeds through a premalignant state of monoclonal gammopathy of unknown significance; however, the molecular events responsible for myelomagenesis remain uncharacterized. To identify cellular pathways deregulated in MM, we addressed that sumoylation is homologous to ubiquitination and results in the attachment of the ubiquitin-like protein Sumo onto target proteins. Sumoylation was markedly enhanced in MM patient lysates compared with normal plasma cells and expression profiling indicated a relative induction of sumoylation pathway genes. The Sumo-conjugating enzyme Ube2I, the Sumo-ligase PIAS1, and the Sumo-inducer ARF were elevated in MM patient samples and cell lines. Survival correlated with expression because 80% of patients with low UBE2I and PIAS1 were living 6 years after transplantation, whereas only 45% of patients with high expression survived 6 years. UBE2I encodes the sole Sumo-conjugating enzyme in mammalian cells and cells transfected with a dominant-negative sumoylation-deficient UBE2I mutant exhibited decreased survival after radiation exposure, impaired adhesion to bone marrow stroma cell and decreased bone marrow stroma cell-induced proliferation. UBE2I confers cells with multiple advantages to promote tumorigenesis and predicts decreased survival when combined with PIAS1. The sumoylation pathway is a novel therapeutic target with implications for existing proteasomal-based treatment strategies.

Activation of NF-kappaB signaling by inhibitor of NF-kappaB kinase beta increases aggressiveness of ovarian cancer.

The NF-kappaB family of transcription factors has been implicated in the propagation of ovarian cancer, but the significance of constitutive NF-kappaB signaling in ovarian cancer is unknown. We hypothesized that constitutive NF-kappaB signaling defines a subset of ovarian cancer susceptible to therapeutic targeting of this pathway. We investigated the biological relevance of NF-kappaB in ovarian cancer using a small-molecule inhibitor of inhibitor of NF-kappaB kinase beta (IKKbeta) and confirmed with RNA interference toward IKKbeta. We developed a gene expression signature of IKKbeta signaling in ovarian cancer using both pharmacologic and genetic manipulation of IKKbeta. The expression of IKKbeta protein itself and the nine-gene ovarian cancer-specific IKKbeta signature were related to poor outcome in independently collected sets of primary ovarian cancers (P = 0.02). IKKbeta signaling in ovarian cancer regulated the transcription of genes involved in a wide range of cellular effects known to increase the aggressive nature of the cells. We functionally validated the effect of IKKbeta signaling on proliferation, invasion, and adhesion. Downregulating IKKbeta activity, either by a small-molecule kinase inhibitor or by short hairpin RNA depletion of IKKbeta, blocked all of these cellular functions, reflecting the negative regulation of the target genes identified. The diversity of functions controlled by IKKbeta in ovarian cancer suggests that therapeutic blockade of this pathway could be efficacious if specific IKKbeta inhibitor therapy is focused to patients whose tumors express a molecular profile suggestive of dependence on IKKbeta activity.The NF-κB family of transcription factors has been implicated in the propagation of ovarian cancer, but the significance of constitutive NF-κB signaling in ovarian cancer is unknown. We hypothesized that constitutive NF-κB signaling defines a subset of ovarian cancer susceptible to therapeutic targeting of this pathway. We investigated the biological relevance of NF-κB in ovarian cancer using a small molecule inhibitor of IKKβ, and confirmed with RNA interference towards IKKβ. We developed a gene expression signature of IKKβ signaling in ovarian cancer using both pharmacologic and genetic manipulation of IKKβ. The expression of IKKβ protein itself and the 9-gene ovarian cancer-specific IKKβ signature were related to poor outcome in independently collected sets of primary ovarian cancers (p=0.02). IKKβ signaling in ovarian cancer regulated the transcription of genes involved in a wide range of cellular effects known to increase the aggressive nature of the cells. We functionally validated the effect of IKKβ signaling on proliferation, invasion and adhesion. Downregulating IKKβ activity, either by a small molecule kinase inhibitor or by shRNA depletion of IKKβ, blocked all of these cellular functions, reflecting the negative regulation of the target genes identified. The diversity of functions controlled by IKKβ in ovarian cancer suggest that therapeutic blockade of this pathway could be efficacious if specific IKKβ inhibitor therapy is focused to patients whose tumors express a molecular profile suggestive of dependence on IKKβ activity.