Andrew Borgman

Cabozantinib (XL184) Inhibits Growth and Invasion of Preclinical TNBC Models

Purpose: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that is associated with poor clinical outcome. There is a vital need for effective targeted therapeutics for TNBC patients, yet treatment strategies are challenged by the significant intertumoral heterogeneity within the TNBC subtype and its surrounding microenvironment. Receptor tyrosine kinases (RTK) are highly expressed in several TNBC subtypes and are promising therapeutic targets. In this study, we targeted the MET receptor, which is highly expressed across several TNBC subtypes. Experimental Design: Using the small-molecule inhibitor cabozantinib (XL184), we examined the efficacy of MET inhibition in preclinical models that recapitulate human TNBC and its microenvironment. To analyze the dynamic interactions between TNBC cells and fibroblasts over time, we utilized a 3D model referred to as MAME (Mammary Architecture and Microenvironment Engineering) with quantitative image analysis. To investigate cabozantinib inhibition in vivo, we used a novel xenograft model that expresses human HGF and supports paracrine MET signaling. Results: XL184 treatment of MAME cultures of MDA-MB-231 and HCC70 cells (± HGF-expressing fibroblasts) was cytotoxic and significantly reduced multicellular invasive outgrowths, even in cultures with HGF-expressing fibroblasts. Treatment with XL184 had no significant effects on METneg breast cancer cell growth. In vivo assays demonstrated that cabozantinib treatment significantly inhibited TNBC growth and metastasis. Conclusions: Using preclinical TNBC models that recapitulate the breast tumor microenvironment, we demonstrate that cabozantinib inhibition is an effective therapeutic strategy in several TNBC subtypes. Clin Cancer Res; 22(4); 923–34. ©2015 AACR.

Partial Deletion of the Sulfate Transporter SLC13A1 Is Associated with an Osteochondrodysplasia in the Miniature Poodle Breed

A crippling dwarfism was first described in the Miniature Poodle in Great Britain in 1956. Here, we resolve the genetic basis of this recessively inherited disorder. A case-control analysis (8∶8) of genotype data from 173 k SNPs revealed a single associated locus on CFA14 (Praw <10–8). All affected dogs were homozygous for an ancestral haplotype consistent with a founder effect and an identical-by-descent mutation. Systematic failure of nine, nearly contiguous SNPs, was observed solely in affected dogs, suggesting a deletion was the causal mutation. A 130-kb deletion was confirmed both by fluorescence in situ hybridization (FISH) analysis and by cloning the physical breakpoints. The mutation was perfectly associated in all cases and obligate heterozygotes. The deletion ablated all but the first exon of SLC13A1, a sodium/sulfate symporter responsible for regulating serum levels of inorganic sulfate. Our results corroborate earlier findings from an Slc13a1 mouse knockout, which resulted in hyposulfatemia and syndromic defects. Interestingly, the metabolic disorder in Miniature Poodles appears to share more clinical signs with a spectrum of human disorders caused by SLC26A2 than with the mouse Slc13a1 model. SLC26A2 is the primary sodium-independent sulfate transporter in cartilage and bone and is important for the sulfation of proteoglycans such as aggregan. We propose that disruption of SLC13A1 in the dog similarly causes undersulfation of proteoglycans in the extracellular matrix (ECM), which impacts the conversion of cartilage to bone. A co-dominant DNA test of the deletion was developed to enable breeders to avoid producing affected dogs and to selectively eliminate the mutation from the gene pool.

Oculoectodermal syndrome is a mosaic RASopathy associated with KRAS alterations.

Oculoectodermal syndrome (OES) is a rare disease characterized by a combination of congenital scalp lesions and ocular dermoids, with additional manifestations including non‐ossifying fibromas and giant cell granulomas of the jaw occurring during the first decade of life. To identify the genetic etiology of OES, we conducted whole‐genome sequencing of several tissues in an affected individual. Comparison of DNA from a non‐ossifying fibroma to blood‐derived DNA allowed identification of a somatic missense alteration in KRAS NM_033360.3(KRAS):c.38G>A, resulting in p.Gly13Asp. This alteration was also observed in the patients other affected tissues including the skin and muscle. Targeted sequencing in a second, unrelated OES patient identified an NM_033360.3(KRAS):c.57G>C, p.Leu19Phe alteration. Allelic frequencies fell below 40% in all tissues examined in both patients, suggesting that OES is a mosaic RAS‐related disorder, or RASopathy. The characteristic findings in OES, including scalp lesions, ocular dermoids, and benign tumors, are found in other mosaic and germline RASopathies. This discovery also broadens our understanding of the spectrum of phenotypes resulting from KRAS alterations. Future research into disease progression with regard to malignancy risk and investigation of RAS‐targeted therapies in OES is warranted. KRAS sequencing is clinically available and may also now improve OES diagnostic criteria.

Single-dose del Nido Cardioplegia in Minimally Invasive Aortic Valve Surgery

del Nido cardioplegia (DC) offers prolonged cardiac protection with single-dose administration and has been shown to be safe in adult CABG surgery. We set out to evaluate the efficacy of cardiac protection and clinical outcomes of DC versus standard blood cardioplegia (BC) in minimally invasive aortic valve surgery. From August 2011 to May 2016, 178 patients underwent minimally invasive aortic valve replacement (mini-AVR) with BC (n = 101) or DC (n = 77). Ministernotomy or right minithoracotomy was utilized for surgical access. Clinical patient characteristics and data were extracted from our local Society of Thoracic Surgeons (STS) database and the electronic medical record. Patients were propensity matched for age, gender, body mass index, valve size and type, STS score, surgical access, preop creatinine, diabetes, and chronic obstructive pulmonary disease, yielding 63 well-matched pairs. There was no difference in patient age, preoperative creatinine, body mass index, diabetes, chronic obstructive pulmonary disease, or STS score between BC and DC before or after propensity matching. BC patients received both anterograde and retrograde cardioplegias in multiple doses, whereas DC was delivered almost entirely anterograde with 95% of the patients (73/77) receiving a single dose only. DC was associated with decreased cardiopulmonary bypass time (108 ± 24 vs 135 ± 43 minutes, P = 0.001) and aortic cross-clamp time (80 ± 16 vs 102 ± 30 min, P = 0.001) and maximal glucose levels during cardiopulmonary bypass (165 ± 39 vs 202 ± 49 mg/dL, P = 0.001), whereas troponin T level did not differ between DC and BC (0.3 ± 0.29 vs 0.44 ± 1.7 ng/mL, P = 0.7). Preoperative ejection fraction did not change in either BC (64% ± 12% vs 61% ± 10%, P = 0.09) or DC (58% ± 14% vs 57% ± 14%, P = 0.4) after AVR. In minimally invasive AVR surgery, DC provided equivalent myocardial protection and clinical outcomes to BC while simplifying cardioprotective regimen and reducing aortic cross-clamp time. DC was associated with lower cardiopulmonary bypass glucose levels and demonstrated the feasibility of a single-dose administration.

WITHDRAWN: Single-dose del Nido cardioplegia in minimally invasive aortic valve surgery

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Abstract A37: Complex crosstalk between MAPK signaling and energy metabolism in melanoma

The notoriously high glucose uptake by metastatic melanoma (MM) lesions is commonly exploited clinically using PET/CT imaging to evaluate disease progression and treatment. The purpose of this study was to use patient-derived xenograft (PDX) MM models and a PDX derived (MM8.1) cell line, to determine how driver oncogene mutations and drugs targeting the RAS-RAF-MEK signaling pathways impact glucose uptake, ATP production and mitochondrial plasticity of MM cells both in vivo and in vitro and the implications on drug resistance. MM BRAF V600E+ patients are routinely treated targeting either mutant BRAF kinase (vemurafenib), or the MAPK downstream signaling intermediate; MEK. To begin exploring links between oncogenic BRAF and metabolism, we first conducted differential gene expression (DGE) profiling of 30 different MM lesions (n=15 patients: pre- and post-engraftment). Focusing on PGC1α; a master mitochondrial regulator promoting oxidative phosphorylation (OXPHOS) revealed lower expression levels in BRAF V600E+ tumors compared to BRAF V600V+ tumors. Further analysis of a BRAF V600E+ MM PDX model revealed strong glucose uptake by PET/CT imaging at baseline. However, 48 hrs. after treatment with vemurafenib triggered growth arrest, accompanied by increased PGC1α; mRNA levels and significant reduction in glucose uptake without change in tumor volume. Upon acquisition of acquired vemurafenib resistance in the PDX model (day 70), rapidly growing tumors displayed reversal to high glucose uptake; which was again diminished by targeting MEK (PD0325901). DGE analysis of vehicle vs. 48 hrs. day 50 and day 70 vemurafenib treated tumors revealed oscillation in the expression of genes regulating glycolysis and oxidative phosphorylation (OXPHOS) that is reflected in the PET/CT imaging. To gain deeper insight, a cell line derived from a vehicle treated tumor, was examined using the Seahorse metabolic analyzer. Vehicle treated MM8.1 cells display high glycolytic capacity and predominantly glycolytic-derived ATP production, which is reflected by only minor inhibition of ATP production with oligomycin (an inhibitor of ATP synthase, a component of the OXHPOS system). Upon treatment with PLX4032, there was rapid reduction in glycolytic capacity along with a concurrent increase in maximum oxygen consumption capacity. PLX4032 treatment additionally enhanced MM8.1 sensitivity to oligomycin, but only transiently (24 hrs. pre-treatment), as later exposure times did not induce oligomycin sensitivity despite DGE changes to indicate otherwise. In conclusion, caution is warranted in evaluating drug responses in patients using PET/CT depending on duration of treatment. Cross-talk involving MAPK signaling and energy metabolism is a dynamic process, with more of a rheostat than switch-type properties, requiring further evaluation to identify therapeutic approaches exploiting metabolic events within MM cells. Citation Format: Nathan J. Lanning, Pimiento Jose, Noel R. Monks, Paula J. Davidson, Andrew S. Borgman, Ting-Tung Chang, Anderson S. Peck, David J. Monsma, Chelsea A. Peterson, Mackeigan P. Jeff, Brian J. Nickoloff. Complex crosstalk between MAPK signaling and energy metabolism in melanoma. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A37. doi: 10.1158/1557-3125.RASONC14-A37

Abstract 361: Illuminating the effects of tissue degradation to improve the management of tissues used in cancer research or clinical applications

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The collection and preservation of tissues from surgery to the lab affects the quality and value of research and/or healthcare applications for cancer patients. To this end we are further defining best practices in the management of surgically resected tissues through the analysis of a systems biology model. In the first steps of this process we defined an experimental animal model to assess gene expression signatures of biological pathways related to preservation intervals. Athymic nude murine tissues (kidney, liver, brain, and PDX-models) were excised at baseline and immediately aliquots of tissue were incubated at varying intervals (t=0, 0.5, 1, 3, 6, 9 hours, temperature during intervals was maintained at 37°C). All tissues were preserved at -80C and total RNA was extracted from these tissues using a uniform RNA isolation kit (RNeasy Mini Kit from Qiagen, Inc.) by the same technician. The RNA Integrity Number (RIN) was obtained using the Agilents BioAnalyzer 2100 and RNA concentration using the Nanodrop 8000. Subsequently, cDNA was synthesized using Thermo Scientifics RevertAid First Strand cDNA Synthesis Kit, and qPCR analysis of each tissue was performed using the Qiagen RT2 Profiler PCR Arrays including the Cell Death PathwayFinder (murine origin liver, kidney, brain) and Hypoxia Signaling Pathway (human origin PDX-model) arrays. Results show a variation of transcripts s that were upregulated (Bax, Bcl2, Fos, Egr1, Tnfrsf10b) or downregulated (Ctss, Hmox1, Epo, Snca) with increased length of tissue incubation and tissue-specific patterns of regulation were observed. Furthermore, these changes in gene expression did not correlate with a decrease in RIN scores, which raises questions about the suitability of RIN as a comprehensive assessment of RNA quality at the transcript level. These findings have important implications for cancer research, namely that tissues that have not been stabilized within several minutes of excision from the host might have undergone degradation of RNA templates that reflect tissue management-related biological activity versus disease-related transcriptome. In future work, we plan to compare macroanalytes such as microRNAs, non-coding RNAs and proteins across different tissue types with an assumption that gene signatures and biological effects will differ per tissue type. We will use results from our experimental biopreservation model to continue the emphasis on transcriptome changes within the context of biological relevance. Citation Format: Scott D. Jewell, Eric Collins, John Beck, David Monsma, Dawna Dylewski, Andrew Borgman, Mary Winn, Galen Hostetter. Illuminating the effects of tissue degradation to improve the management of tissues used in cancer research or clinical applications. [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 361. doi:10.1158/1538-7445.AM2014-361

Abstract 1213: Comparing protein pathway activation mapping portraits between gliobastoma patient-matched primary tumor, xenografts and neurospheres: implications for precision medicine

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Most current targeted therapeutics are directed against the activated protein kinase-driven signaling pathway architecture. While guiding therapy selection based on protein phosphorylation is biochemically rational given these proteins represent the direct drug targets it is not certain which protein networks/signaling protein targets are the causal driver of any patients tumor. Thus, the availability of a system that recapitulates the human in vivo setting and allows for credentialing and identification of which activated signaling networks are causally necessary for tumor viability would be extremely important. Methods: To test two such systems and determine how they resemble the cell signaling architecture of the human primary tumor we compared eight snap-frozen laser capture microdissected (LCM) primary glioblastoma multiforme (GBM) samples with tumor-matching LCM mouse xenografts as well as matching neurospheres (NP). Reverse phase protein microarrays were utilized wherein the total level or phosphorylated level of 70 key signal transduction proteins known to be the direct targets for a number of important targeted therapies and implicated in GBM tumor biology were measured. Results: Overall, the signaling architecture of the xenografts more closely resembled the matched primary LCM tumors in this study set. However, unsupervised hierarchical clustering of the data revealed instances where pathway information such as total c-MET, phospho RAF (S259), phospho GSK3αβ (S21/9) and total EGFR was retained across matched LCM primary tumor, xenografts and NP. In addition there were a number of instances where the signaling architecture of the primary human tumor did not resemble the matched NP and xenograft signature. Conclusion: These pilot data indicate that 3D cell culture conditions may effectively recapitulate in vivo signaling network activation in certain instances, although these preliminary results also suggest that xenografts more closely resemble the cell architecture of matching primary GBM tumors. These data highlight the importance and impact of the microenvironment and culture conditions on tumor cell signal transduction network activation. However, two caveats to this approach remain: (a) drug studies in GBM xenografts and NP are limited to those targeting signaling pathways that are faithfully reproduced from the human in vivo setting and (b) the time required to develop a patient matched xenograft model for individualized drug testing may be too long for a fast-progressing disease such as GBM. Citation Format: Claudius Mueller, Ana C. deCarvalho, Tom Mikkelsen, Laila Poisson, Valerie Calvert, Andrew Borgman, David M. Cherba, Mary E. Winn, Emanuel F. Petricoin. Comparing protein pathway activation mapping portraits between gliobastoma patient-matched primary tumor, xenografts and neurospheres: implications for precision medicine. [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 1213. doi:10.1158/1538-7445.AM2014-1213

Abstract B12: Overcoming acquired resistance to vemurafenib using clinically relevant PDX models of melanoma.

Development of resistance is a significant clinical problem for virtually all targeted cancer therapies. We have generated a reproducible, patient derived xenograft (PDX) model of acquired vemurafenib resistance to address these challenges. Continuous treatment of V600E melanoma tumors, caused synchronous tumor stasis for approximately 7 weeks, following which, all tumors displayed simultaneous resistance marked by rapid tumor growth. Additionally, this model maintains the resistance phenotype upon serial transplantation, providing a platform for testing rational drug selection. The fidelity of the PDX models was further confirmed using a BRAF V600V tumor which did not respond to vemurafenib. Onset of vemurafenib resistance is accompanied by increased phosphor-ERK signifying re-engagement of the MAPK signaling pathway and supporting MEK as a potential target. MEK inhibition in vemurafenib resistant tumors using PD0325901, resulted in rapid tumor shrinkage and dramatically reduced phosphor-ERK levels. Treatment of resistant tumors with PD0325901 alone, whilst leading to rapid tumor shrinkage, showed significant host toxicity and onset of acquired MEKi resistance. Interestingly, combination of vemurafenib + PD0325901 was non-toxic, and showed dramatic and sustained tumor suppression. Upon cessation of PD0325901 at 70 days the tumors remained undetectable for the duration of the study (>100 days). These data support the use of MEK inhibitors post-development of vemurafenib resistance and demonstrate that combination therapy mitigates systemic MEKi toxicity and results in persistent tumor inhibition/eradication. PDX models of acquired resistance provide a unique opportunity to bridge the gap between patients and the basic in vitro biology. Additionally, this PDX system allows the interrogation of the kinetics involved in the development of resistance by longitudinal tumor tissue sampling. Numerous mechanisms have been identified as potential causes of the resistance phenotype. Many have been identified in vitro but not all have been confirmed in patients. We detected no evidence of increased BRAF copy number or expression, although alternative BRAF splicing was identified in resistant tumors. Using differential gene expression accompanied by pathway and network analysis we identified distinct differences in the PDX tumors at various time points during the development of resistance. In particular, a potential role for interferon signaling in resistant tumors was observed. Furthermore, changes in the metabolic profiles were identified with untreated and resistant tumors favoring glycolytic pathways, whereas growth arrested tumors exhibited a preference for oxidative phosphorylation. In conclusion, these results demonstrate the value of PDX models for contributing to clinical cancer management through the decryption of complex drug resistance mechanisms and accelerating the identification of rationally selected drug combinations for bench to bedside applications. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B12. Citation Format: Noel R. Monks, David J. Monsma, David M. Cherba, Emily Eugster, Dawna Dylewski, Mary E. Winn, Andrew S. Borgman, Paula J. Davidson, Chelsea A. Peterson, Jose M. Pimiento, Alexander E. Ivliev, Yuri Nikolsky, Marina Bessarabova, Valerie S. Calvert, Mariaelena Pierobon, Emanuel F. Petricoin, Craig P. Webb, Brian J. Nickoloff. Overcoming acquired resistance to vemurafenib using clinically relevant PDX models of melanoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B12.