Patricia Burke

Significant antitumor activity in vivo following treatment with the microtubule agent ENMD-1198

Clinical studies using the microtubule-targeting agent 2-methoxyestradiol (2ME2; Panzem) in cancer patients show that treatment is associated with clinical benefit, including prolonged stable disease, complete and partial responses, and an excellent safety profile. Studies have shown that 2ME2 is metabolized by conjugation at positions 3 and 17 and oxidation at position 17. To define structure-activity relationships for these positions of 2ME2 and to generate metabolically stable analogues with improved anti-tubulin properties, a series of analogues was generated and three lead analogues were selected, ENMD-1198, ENMD-1200, and ENMD-1237. These molecules showed improved metabolic stability with >65% remaining after 2-h incubation with hepatocytes. Pharmacokinetic studies showed that oral administration of the compounds resulted in increased plasma levels compared with 2ME2. All three analogues bind the colchicine binding site of tubulin, induce G2-M cell cycle arrest and apoptosis, and reduce hypoxia-inducible factor-1α levels. ENMD-1198 and ENMD-1200 showed improved in vitro antiproliferative activities. Significant reductions in tumor volumes compared with vehicle-treated mice were observed in an orthotopic breast carcinoma (MDA-MB-231) xenograft model following daily oral treatment with all compounds (ANOVA, P < 0.05). Significantly improved median survival time was observed with ENMD-1198 and ENMD-1237 (200 mg/kg/d) in a Lewis lung carcinoma metastatic model (P < 0.05). In both tumor models, the high-dose group of ENMD-1198 showed antitumor activity equivalent to that of cyclophosphamide. ENMD-1198 was selected as the lead molecule in this analogue series and is currently in a phase I clinical trial in patients with refractory solid tumors. [Mol Cancer Ther 2008;7(6):1472–82]

Phase I Dose-Escalation Study of MEDI-573, a Bispecific, Antiligand Monoclonal Antibody against IGFI and IGFII, in Patients with Advanced Solid Tumors

Purpose: This phase I, multicenter, open-label, single-arm, dose-escalation, and dose-expansion study evaluated the safety, tolerability, and antitumor activity of MEDI-573 in adults with advanced solid tumors refractory to standard therapy or for which no standard therapy exists. Experimental Design: Patients received MEDI-573 in 1 of 5 cohorts (0.5, 1.5, 5, 10, or 15 mg/kg) dosed weekly or 1 of 2 cohorts (30 or 45 mg/kg) dosed every 3 weeks. Primary end points included the MEDI-573 safety profile, maximum tolerated dose (MTD), and optimal biologic dose (OBD). Secondary end points included MEDI-573 pharmacokinetics (PK), pharmacodynamics, immunogenicity, and antitumor activity. Results: In total, 43 patients (20 with urothelial cancer) received MEDI-573. No dose-limiting toxicities were identified, and only 1 patient experienced hyperglycemia related to treatment. Elevations in levels of insulin and/or growth hormone were not observed. Adverse events observed in >10% of patients included fatigue, anorexia, nausea, diarrhea, and anemia. PK evaluation demonstrated that levels of MEDI-573 increased with dose at all dose levels tested. At doses >5 mg/kg, circulating levels of insulin-like growth factor (IGF)-I and IGFII were fully suppressed. Of 39 patients evaluable for response, none experienced partial or complete response and 13 had stable disease as best response. Conclusions: The MTD of MEDI-573 was not reached. The OBD was 5 mg/kg weekly or 30 or 45 mg/kg every 3 weeks. MEDI-573 showed preliminary antitumor activity in a heavily pretreated population and had a favorable tolerability profile, with no notable perturbations in metabolic homeostasis. Clin Cancer Res; 20(18); 4747–57. ©2014 AACR.

The hematopoietic-specific β1-tubulin is naturally resistant to 2-Methoxyestradiol and protects patients from drug-induced myelosuppression

Taxanes and other microtubule-targeting drugs (MTDs) represent one of the most effective classes of cancer chemotherapeutics. However, ultimately their utility is limited due to drug-induced myelosuppression. Here we identify 2-Methoxyestradiol (2ME2) as the first MTD able to specifically target tumor cells while sparing the bone marrow from dose-limiting, life-threatening toxicities. Following drug selection with 2ME2, epithelial cancer cells acquired a tubulin mutation at Vβ236I that impaired the 2ME2-tubulin interaction and rendered cells resistant to 2ME2. We further show that the hematopoietic-specific Hβ1 tubulin isotype naturally encodes Iβ236 and is insensitive to 2ME2. Systemic administration of 2ME2 in C57BL6 mice revealed that there was no effect on bone marrow microtubules, in contrast to the taxane or Vinca alkaloid induced toxicities. Similar results were obtained upon drug treatment of human bone marrow and CD34-positive stem/progenitor cells. Herein, we describe the first isotype-targeted chemotherapeutic, setting a new paradigm for the entire class of MTDs, and providing a model that could allow the design of new tubulin inhibitors devoid of myelosuppression. The ability to design a drug with minimal side-effects would significantly augment the chances of clinical success by allowing the use of a truly therapeutic dose rather than the maximally tolerated.

Circulating Tumor Cells: Application as a Biomarker for Molecular Characterization and Predictor of Survival in an All-Comer Solid Tumor Phase I Clinical Study

Purpose Clinical development of cancer drugs has a low success rate. Prognostic and predictive biomarkers using minimally invasive approaches hold promise for increasing the probability of success by enabling disease characterization, patient selection and early detection of drug treatment effect. Enumeration and molecular characterization of circulating tumor cells (CTC) may address some of these needs, and thus were evaluated for utility in a Phase I solid tumor clinical study. Experimental Design Blood samples for CTC analysis were obtained from 24 cancer patients in a multi-center all-comer Phase I study of MEDI-575, a novel anti-PDGFRα antibody. Samples were taken at screening and analyzed for enumeration of CTC using the CellSearch® platform and for molecular characterization using a novel quantitative RT-PCR assay. Results Fifty-nine percent of the patients showed at least 1 CTC per 7.5 ml of blood at baseline. Progression-free survival (PFS) and overall survival (OS) of patients with 0 CTCs at baseline were longer than PFS and Os for patients with 1-3 and >3 CTCs (8.8 versus 1.4 and 1.3 months PFS, P = 0.02; 9.0 vs 7.4 and 3.5 months OS, P = 0.20, respectively). Patients with 0 CTC showed a greater percentage of stable disease than the other 2 groups with 1-3 and >3 CTCs (57% vs 29% and 0%). The multimarker qRT-PCR method detected CTC in 40% of the patients, and 80% of these patients were positive for pre-selected drug target genes. Conclusion CTC enumeration of patients in an all-comer study is feasible and may allow for patient stratification for PFS and Os to evaluate the clinical response of investigational agents. Gene expression profiling of isolated CTC may provide a means for molecular characterization of selected tumor targets.

Characterization of the anti‐CD22 targeted therapy, moxetumomab pasudotox, for B‐cell precursor acute lymphoblastic leukemia

Moxetumomab pasudotox is a second‐generation recombinant immunotoxin against CD22 on B‐cell lineages. Antileukemic activity has been demonstrated in children with chemotherapy‐refractory B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL), with variable responses. Here, we report in vitro and in vivo evaluation of moxetumomab pasudotox treatment of human cell lines and patient‐derived cells as a preliminary study to understand characteristics of sensitivity to treatment. Binding, internalization, and apoptosis were evaluated using fluorescently tagged moxetumomab pasudotox. Studies in NOD‐scid IL2Rgnull mice showed a modest survival benefit in mice engrafted with 697 cells but not in NALM6 or the two patient‐derived xenograft models.

Abstract 4420: Factors potentially contributing to sensitivities of CD22-targeting agents in B-cell malignancies

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA In order to understand factors which may contribute to in vitro sensitivity of B-cell malignancies to CD22-targeting agents, we evaluated the mRNA expression levels of genes involved with diphthamide biosynthesis, multi-drug resistance, and CD22 (including the splice variant 3 with exon12 deletion) in untreated and 72-hour CD22-targeting agent-treated DLBCL and CLL cell lines. Cell line sensitivity (EC50) to CD22-targeting agent treatment was evaluated by 72 hour exposureto moxetumomab pasudotox (MP).Cell lines were characterized as insensitive if the EC50s were greater than100 ng/mL. The sensitivity to CD22 -targeting agent treatment was found to be significantly associated with baseline CD22 mRNA expression level, as determined by regression analysis. The highly resistant DLBCL cell line RCK8 and the resistant CLL cell line Z138 demonstrated low CD22 expression, corresponding to their elevated EC50′s. However, baseline mRNA was not significantly lower in some other less sensitive cell lines, suggesting that total mRNA CD22 levels may not be the only factor contributing to reduced sensitivity to anti-CD22 exposure. Given the critical roles of dipthamides in CD22 targeting, we evaluated baseline DPH4 levels and their methylation status. There was no correlation between CD22-targeting agent sensitivity and baseline levels of DPH4 expression/promoter methylation. We further explored how CD22, DPH4, WDR85 and MRP1 mRNA expression changed following a 72 hour exposure to MP. Following MP exposure, both mRNA and methylation levels of DPH4 were not significantly changed. However, the mRNA levels of both the diphthamide biosynthesis protein WDR85 and multidrug resistance-associated protein 1 (MRP1) were up-regulated in half of the cell lines following MP exposure. As expression of truncated CD22 has been reported in ALL, we also evaluated CD22 variant expression before and following CD22-targeting agent exposure. All CD22 variants were co-expressed in most cell lines, with significantly increased V3 observed following treatment in some cell lines with lower EC50′s. In conclusion, our results show sensitivity to CD22-targeting agent in the majority of the cell lines tested, and suggest that multiple factors may contribute to sensitivity including CD22 expression. Additionally, our results suggest that factors which may affect continued response to CD22 targeting agents may change with treatment, including increases in CD22 variant 3, MRP1 and WDR85. Ongoing research will evaluate whether these changes are relevant to CD22-targeting agent sensitivity in vivo. Citation Format: Xin Yao, Patricia Burke, Joyce O. Obidi, Xiaoru Chen, Haifeng Bao, Yihong Yao, Jiaqi Huang. Factors potentially contributing to sensitivities of CD22-targeting agents in B-cell malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4420. doi:10.1158/1538-7445.AM2015-4420

Abstract 4501: Sensitivities of kinase inhibitors of BCR signaling are correlated with the BCR signaling pathway activities in DLBCL and CLL

B-cell malignancies are hematologic disorders for which new therapeutic agents and novel treatment strategies are evolving. B cell receptor (BCR) signaling plays an important pathogenic role in diffuse large b-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL). There is rapid clinical development of inhibitors targeting BCR-associated kinases such as Spleen tyrosine kinase (SYK), Bruton9s tyrosine kinase (BTK), and phosphoinositide 3-kinase (PI3K)δ. Recent clinical studies using these agents have demonstrated effectiveness and low toxicity in CLL and DLBCL patients with relapsed/refractory disease. With a great response rate, there are still a group of patients that do not respond to these therapeutic agents. The goal of this study is to investigate potential resistance mechanism(s) of BCR pathway inhibitors based on signature-signaling profiles. 21 CLL and DLBCL cell lines with different molecular features were assessed in this study to establish relevant signaling features and their association with sensitivities to BCR signaling inhibitors. First, we evaluated response of cell lines to small molecule inhibitors targeting the BCR signaling pathways (PCI-32765, R406, CAL 101, and Dasatinib). 28% (6/21) of the cell lines were resistant to PCI-32765; we found that these cells lines were also non responsive to CAL 101 and Dasatinib. Then we used phosphoflow assays and phosphoprotein arrays to profile BCR signaling in both responsive and non-responsive cell lines. The BCR signaling activities were assessed at baseline, after stimulation with IgG-IgM, and following pretreatment with phosphatase inhibitors. Interrogating pathway activity in cell lines with pervanadate, a phosphatase inhibitor, was especially revealing. After treatment with pervanadate, we observed an increase in phosphorylation status of SYK, BTK, p38, and S6 in normal B cells. However, 24% of tumor cell lines do not exhibit phosphorylation of BCR related proteins, SYK and BTK, after pervanadate treatment. Moreover, 83% (5/6) of cell lines resistant to PCI 32765 (IC50 > 20μM) did not respond to pervanadate, whereas only 12.5% (1/8) sensitive cell lines (IC50 Citation Format: Joyce O. Obidi, Patricia Burke, Laura Richman, Dirk Mendel, Haifeng Bao. Sensitivities of kinase inhibitors of BCR signaling are correlated with the BCR signaling pathway activities in DLBCL and CLL. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4501. doi:10.1158/1538-7445.AM2014-4501

Abstract 2425: Nucleosomal DNA assay development and utility as PoP biomarker.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Elevated baseline levels of circulating cell-free nucleosomal DNA (nDNA) are observed in some cancer patients and may be associated with clinical outcome and tumor burden. Transient increases in nDNA in response to effective chemotherapy have been observed, indicating nDNA could be a biomarker for apoptosis and could serve as a minimally invasive proof of principal (PoP) biomarker for cancer treatments. The Cell Death Detection ELISA has been used to measure nDNA levels, but the non-quantitative nature of this assay greatly limits its application in clinical studies. Our objectives were to develop a qualified assay for semi-quantitative measurement of nDNA levels in patient plasma samples and utilize the assay in Phase 1 clinical trials to assess its utility. Here we report on nDNA assay development and its application in 2 clinical trials in hematological malignancies. In order to develop a more quantitative assay, we made nDNA standards from healthy-donor blood samples and established their stability using lyophilized plasma. Recovery of freeze-thawed nDNA standard and plasma samples from normal donors and cancer patients were performed. Methods were established for the collection, storage, and analysis of plasma samples. Samples from subjects in Phase 1 dose-escalation studies undergoing treatment with MEDI-551, an anti-CD19 ADCC-enhanced monoclonal antibody (25 subjects, 0.5 - 12 mg/Kg) or moxetumomab pasudotox (CAT-8015), an anti-CD22 immunotoxin (23 subjects, 20 - 60 μg/Kg) were collected at various time points to assess changes in nDNA with treatment. Predose samples were used to assess baseline levels of nDNA for normalizing responses, comparing differences by type of malignancy, and for prognostic purposes. We developed a semi-quantitative, highly reproducible assay with low intra- and inter-plate variability. Although initial sample thaws significantly decreased signal recovery, subsequent thaws did not. Baseline levels of patient nDNA samples were not significantly different across tumor types (DLBCL, FL, CLL, MCL, MM; P=0.1170). The time course of nDNA changes in the first cycle of treatment illustrated potential differences in mechanism of action and dosing for the 2 studies, with short pronounced fold change (FC) increases occurring early in the cycle (Day 2) for MEDI-551 (weekly dosing, cycle 1) compared to prolonged FC increases at Day 5 for CAT-8015 (dosed day 1, 3, 5 every 28 days). A trend of increased nDNA with dose (MEDI-551, P=0.1522; CAT-8015, P=0.2087) and significantly increased FC for end of treatment samples was noted for both studies (MEDI-551, P=0.0438; CAT-8015, P=0.0014). These results indicate that FC increases of nDNA in response to treatment could potentially provide a proof of principle (PoP) biomarker in some clinical oncology studies. In conclusion, we have developed a new semi-quantitative qualified assay for assessing nDNA in blood which has been utilized to support clinical drug development. Citation Format: Xiaoqing (Sarah) Shi, Haifeng Bao, Yuling Wu, Xiaoru Chen, David L. Gold, Theresa M. LaVallee, Patricia A. Burke. Nucleosomal DNA assay development and utility as PoP biomarker. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2425. doi:10.1158/1538-7445.AM2013-2425

Abstract 5582: Interrogation of signaling networks in DLBCL to improve classification of patient subsets for targeted therapy.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Diffuse large B cell lymphoma (DLBCL) is the most common B cell malignancies in adults. Molecular classification of DLBCL based on gene expression profiles has demonstrated prognostic value in predicting patient outcome. While, the classification of DLBCL based on gene expression profiles provides some information on signaling pathways, analysis at the protein level may also help to inform treatment for DLBCL patients. The objectives of this study were: 1) to identify signature-cell signaling profiles in DLBCL and 2) to classify DLBCL based on cell signaling profiles that are associated with response to therapeutic agents. Several of DLBCL cell lines with different molecular features were assessed in this study. The cell signaling activities of MAPK, AKT, and NFκB pathways were interrogated using phospho-flow technology in these cell lines at baseline or after activation by external stimulation. We observed that MAPK and AKT signaling in DLBCL cell lines contrasted significantly with signaling in normal B cells in response to ex vivo stimulation; DLBCL cells lines show constitutive activation of the MAPK and AKT signaling pathways at baseline. The ABC DLBCL cell line, U2932, showed enhanced MAPK and AKT signaling after BCR engagement (αBCR). In the case of NFκB signaling, ex vivo stimulation reveals an absence of functional NFκB activity in the GCB cell lines. The lack of NFκB pathway activity observed in GCB cell lines was restored with the use of a phosphatase inhibitor, indicating that impaired signaling is not through loss of kinase function but is due to an increase in negative regulation. The inconsistency in MAPK, AKT, and NFκB pathway activities could possibly aid in defining a signaling profile applicable to patient stratification. Furthermore, this phospho-flow signaling data has provided an opportunity to refine the signaling model specifically in the case of αBCR. Following cross-linking of the BCR receptor, we observed no phosphorylation of BCR network proteins (pERK and pS6) in normal B cells which contrasted with higher phosphorylation of these proteins observed in cell lines with an ABC phenotype. Additionally, the sensitivities of these DLBCL cell lines to various chemotherapeutic agents were also assessed. The relationship between cell signaling signatures, drug sensitivity, and gene mutational status are being analyzed. Overall, our results suggest that profiling of signaling network activities represents a promising approach to molecularly classify DLBCL and warrants further investigation in a clinical setting. Citation Format: Joyce O. Obidi, Patricia Burke, Serguei Soukharev, Laura Richman, Dirk Mendel, Theresa LaVallee, Haifeng Bao. Interrogation of signaling networks in DLBCL to improve classification of patient subsets for targeted therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5582. doi:10.1158/1538-7445.AM2013-5582

Abstract 3484: CTC enumeration and molecular characterization in a Phase 1 study of IGF-1 and IGF-2 inhibition by MEDI-573.

MEDI-573 selectively binds to human insulin-like growth factor (IGF)-1 and IGF-2 and inhibits ligand-mediated signal transduction via the insulin-like growth factor 1 receptor (IGF-1R) and insulin receptor A isoform (IR-A) pathways, without perturbing glucose homeostasis. As IGF-1, IGF-2, and IGF-1R are overexpressed in bladder cancer, a Phase 1 expansion study including 20 bladder cancer subjects along with 6 additional subjects with other types of cancers from the dose escalation study were included for circulating tumor cell (CTC) analysis as part of a larger 42-subject study (MI-CP184) evaluating PK, safety, and biomarkers. Our objective for the results reported here was to evaluate CTC numbers as a prognostic indicator and as a biomarker of response to MEDI-573 therapy, and to evaluate the expression of IGF-1R on CTC and any correlation with treatment. Subjects in the expansion study were treated with MEDI-573 at 5 mg/Kg weekly (QW) (n=10), 15 mg/Kg QW (n=10), whereas subjects evaluated for CTC in the dose escalation study were treated at 30 or 45 mg/Kg every 3 weeks (Q3W) (n=3 each). CTC collected at screening and pre-dosing each cycle were enumerated and evaluated for IGF-1R expression by CellSearch®. 92% of subjects had at least one CTC at any time during the study and 62.5% had at least one CTC at screening. As has been demonstrated with other tumor types, subjects with ≤3 CTC at screening stayed on study longer than subjects with >3 CTC (2.3 vs. 1.2 months, P=0.0125 [no censoring]). Mean overall survival (OS) was also longer for subjects with ≤3 CTC compared to subjects with >3 CTC (6.37 vs. 2.83 months (P=0.0218, t test), although Kaplan-Myer curves were not different (P=0.0854)). Subjects with decreased numbers of CTC or no change in CTC with treatment stayed on study longer (not significant, P=0.0512, t test) than subjects with increased CTC with treatment (2.95 vs. 1.70 months). 73.3% (11/15) of subjects with positive CTC at screening had at least 1 CTC positive for IGF-1R. In conclusion, our results provide evidence for the utility of CTC as prognostic indicators in a Phase 1 study. Citation Format: Xiaoru Chen, Xiaoqing (Sarah) Shi, Haifeng Bao, Theresa M. LaVallee, Dirk B. Mendel, Patricia A. Burke. CTC enumeration and molecular characterization in a Phase 1 study of IGF-1 and IGF-2 inhibition by MEDI-573. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3484. doi:10.1158/1538-7445.AM2013-3484