Melanie Galvin

Early Prediction of Polymyxin-Induced Nephrotoxicity With Next-Generation Urinary Kidney Injury Biomarkers

Despite six decades of clinical experience with the polymyxin class of antibiotics, their dose-limiting nephrotoxicity remains difficult to predict due to a paucity of sensitive biomarkers. Here, we evaluate the performance of standard of care and next-generation biomarkers of renal injury in the detection and monitoring of polymyxin-induced acute kidney injury in male Han Wistar rats using colistin (polymyxin E) and a polymyxin B (PMB) derivative with reduced nephrotoxicity, PMB nonapeptide (PMBN). This study provides the first histopathological and biomarker analysis of PMBN, an important test of the hypothesis that fatty acid modifications and charge reductions in polymyxins can reduce their nephrotoxicity. The results indicate that alterations in a panel of urinary kidney injury biomarkers can be used to monitor histopathological injury, with Kim-1 and α-GST emerging as the most sensitive biomarkers outperforming clinical standards of care, serum or plasma creatinine and blood urea nitrogen. To enable the prediction of polymyxin-induced nephrotoxicity, an in vitro cytotoxicity assay was employed using human proximal tubule epithelial cells (HK-2). Cytotoxicity data in these HK-2 cells correlated with the renal toxicity detected via safety biomarker data and histopathological evaluation, suggesting that in vitro and in vivo methods can be incorporated within a screening cascade to prioritize polymyxin class analogs with more favorable renal toxicity profiles.

Tumourigenic non-small-cell lung cancer mesenchymal circulating tumour cells: a clinical case study

An explant model derived from EpCam negative mesenchymal non-small-cell lung (NSCLC) cancer circulating tumour cells (a ‘liquid biopsy’) recapitulates the histology of the donor patients diagnostic specimen and chemoresistance to cisplatin and pemetrexed. This proof-of-principal landmark model opens a new avenue for study of advanced NSCLC biology when tissue biopsies unavailable.

Inhibition of PI3K/BMX Cell Survival Pathway Sensitizes to BH3 Mimetics in SCLC.

Most small cell lung cancer (SCLC) patients are initially responsive to cytotoxic chemotherapy, but almost all undergo fatal relapse with progressive disease, highlighting an urgent need for improved therapies and better patient outcomes in this disease. The proapoptotic BH3 mimetic ABT-737 that targets BCL-2 family proteins demonstrated good single-agent efficacy in preclinical SCLC models. However, so far clinical trials of the BH3 mimetic Navitoclax have been disappointing. We previously demonstrated that inhibition of a PI3K/BMX cell survival signaling pathway sensitized colorectal cancer cells to ABT-737. Here, we show that SCLC cell lines, which express high levels of BMX, become sensitized to ABT-737 upon inhibition of PI3K in vitro, and this is dependent on inhibition of the PI3K-BMX-AKT/mTOR signaling pathway. Consistent with these cell line data, when combined with Navitoclax, PI3K inhibition suppressed tumor growth in both an established SCLC xenograft model and in a newly established circulating tumor cell–derived explant (CDX) model generated from a blood sample obtained at presentation from a chemorefractory SCLC patient. These data show for the first time that a PI3K/BMX signaling pathway plays a role in SCLC cell survival and that a BH3 mimetic plus PI3K inhibition causes prolonged tumor regression in a chemorefractory SCLC patient–derived model in vivo. These data add to a body of evidence that this combination should move toward the clinic. Mol Cancer Ther; 15(6); 1248–60. ©2016 AACR.

PDGFR-modulated miR-23b cluster and miR-125a-5p suppress lung tumorigenesis by targeting multiple components of KRAS and NF-kB pathways.

In NSCLC alterations in PDGF receptors are markers of worst prognosis and efficient targeting of these receptors is yet to be achieved. In this study, we explored PDGFR-regulated microRNAs demonstrating that miR-23b cluster and miR-125a-5p are downregulated by increased expression of PDGFR-α or PDGFR-β in NSCLC cells. Mechanistically, the expression of these microRNAs is positively regulated by p53 and negatively modulated by NF-kB p65. Forced expression of miR-23b cluster or miR-125a-5p enhanced drug sensitivity and suppressed invasiveness of NSCLC cells by silencing several genes involved in oncogenic KRAS and NF-kB pathways, including SOS1, GRB2, IQGAP1, RALA, RAF-1, IKKβ, AKT2, ERK2 and KRAS itself. Of note, an inverse correlation between miR-23b cluster, miR-125a-5p and respective target genes was also found in vivo in a large dataset of lung adenocarcinoma samples. Furthermore, in vivo delivery of miR-23b cluster or miR-125a-5p significantly repressed tumour growth in a highly aggressive NSCLC circulating tumour cell (CTC) patient derived explant (CDX) mouse model. In conclusion, our finding sheds light on the PDGFR signaling and endorses the possibility to employ miR-23b cluster and miR-125a-5p as therapeutic tools to silence simultaneously a range of redundant pathways and main effectors of tumorigenesis in NSCLC.

The Combination of the PARP Inhibitor Olaparib and the WEE1 Inhibitor AZD1775 as a New Therapeutic Option for Small Cell Lung Cancer

Purpose: Introduced in 1987, platinum-based chemotherapy remains standard of care for small cell lung cancer (SCLC), a most aggressive, recalcitrant tumor. Prominent barriers to progress are paucity of tumor tissue to identify drug targets and patient-relevant models to interrogate novel therapies. Following our development of circulating tumor cell patient–derived explants (CDX) as models that faithfully mirror patient disease, here we exploit CDX to examine new therapeutic options for SCLC. Experimental Design: We investigated the efficacy of the PARP inhibitor olaparib alone or in combination with the WEE1 kinase inhibitor AZD1775 in 10 phenotypically distinct SCLC CDX in vivo and/or ex vivo. These CDX represent chemosensitive and chemorefractory disease including the first reported paired CDX generated longitudinally before treatment and upon disease progression. Results: There was a heterogeneous depth and duration of response to olaparib/AZD1775 that diminished when tested at disease progression. However, efficacy of this combination consistently exceeded that of cisplatin/etoposide, with cures in one CDX model. Genomic and protein analyses revealed defects in homologous recombination repair genes and oncogenes that induce replication stress (such as MYC family members), predisposed CDX to combined olaparib/AZD1775 sensitivity, although universal predictors of response were not noted. Conclusions: These preclinical data provide a strong rationale to trial this combination in the clinic informed by prevalent, readily accessed circulating tumor cell–based biomarkers. New therapies will be evaluated in SCLC patients after first-line chemotherapy, and our data suggest that the combination of olaparib/AZD1775 should be used as early as possible and before disease relapse. Clin Cancer Res; 24(20); 5153–64. ©2018 AACR.

Signaling pathway screening platforms are an efficient approach to identify therapeutic targets in cancers that lack known driver mutations: a case report for a cancer of unknown primary origin

Precision medicine aims to tailor cancer therapies to target specific tumor-promoting aberrations. For tumors that lack actionable drivers, which occurs frequently in the clinic, extensive molecular characterization and pre-clinical drug efficacy studies will be required. A cell line maintained at low passage and a patient- derived xenograft model (PDX) were generated using a fresh biopsy from a patient with a poorly-differentiated neuroendocrine tumor of unknown primary origin. Next-generation sequencing, high throughput signaling network analysis, and drug efficacy trials were then conducted to identify actionable targets for therapeutic intervention. No actionable mutations were identified after whole exome sequencing of the patient’s DNA. However, whole genome sequencing revealed amplification of the 3q and 5p chromosomal arms, that include the PIK3CA and RICTOR genes, respectively. We then conducted pathway analysis, which revealed activation of the AKT pathway. Based on this analysis, efficacy of PIK3CA and AKT inhibitors were evaluated in the tumor biopsy-derived cell culture and PDX, and response to the AKT inhibitor AZD5363 was observed both in vitro and in vivo indicating the patient would benefit from targeted therapies directed against the serine/threonine kinase AKT. In conclusion, our study demonstrates that high throughput signaling pathway analysis will significantly aid in identifying actionable alterations in rare tumors and guide patient stratification into early-phase clinical trials.

Signaling pathway screening platforms are an efficient approach to identify therapeutic targets in precision-medicine oriented early phase clinical trials

Precision medicine aims to tailor cancer therapies to target specific tumorpromoting aberrations. For tumors that lack actionable drivers, extensive molecular characterization and pre-clinical drug efficacy studies will be required to match patients with the appropriate targeted therapy. A cell line maintained at low passage and a patient-derived xenograft model (PDX) were generated using a fresh biopsy from a patient with a poorly-differentiated neuroendocrine tumor of unknown primary origin. Next-generation sequencing, high throughput signaling network analysis, and drug efficacy trials were then conducted to identify actionable targets for therapeutic intervention. No actionable mutations were identified after whole exome sequencing of the patient’s DNA; however, whole genome sequencing revealed amplification of the 3q and 5p chromosomal arms, that include the PIK3CA and RICTOR genes, respectively. Consistent with amplification of these genes, pathway analysis revealed activation of the AKT pathway. Based on this analysis, efficacy of PIK3CA and AKT inhibitors were evaluated in the tumor biopsy-derived cell culture and PDX, and response to the AKT inhibitor AZD5363 was observed both in vitro and in vivo indicating the patient would benefit from targeted therapies directed against the serine/threonine kinase AKT. In conclusion, our study demonstrates that high throughput signaling pathway analysis complements next-generation sequencing approaches for detection of actionable alterations and will aid in patient stratification into early-phase clinical trials.

019 Chemotherapy-induced cardiotoxicity: could a translational cardiac MRI model help identify patients at risk?

Background Cancer survivorship is an international priority and mortality from cardiac damage is one of the greatest concerns. Anthracycline chemotherapy is cardiotoxic but remains highly effective against cancer. Methods This translational project involved the development of a pre-clinical rat model of progressive anthracycline-induced cardiotoxicity (1.25mg/m2 doxorubicin weekly for 8 doses) and a concurrent clinical study of 30 cancer patients receiving 6 cycles of curative anthracycline therapy (doxorubicin 50mg/m2). A bespoke cardiac magnetic resonance imaging (CMR) protocol was developed which included longitudinal T1 mapping (modified look-locker) to estimate extracellular volume (ECV), T2 mapping (to quantify oedema) and strain analysis. A panel of circulating biomarkers (including inflammatory, ischaemic and fibrosis markers) was evaluated by multiplex ELISA. Longitudinal histological analysis was performed in the rats and correlated with imaging and biomarker findings. Results Left ventricular function (LVEF) declined steadily during treatment in rats and humans. Persistent LV dysfunction was seen in 23% of patients 12 months after therapy. Peak Troponin I levels correlated with fall in (LVEF) and circulating levels translated well between rat and humans. However, levels did not peak until the final cycle of chemotherapy when significant LV decline had already occurred. There was no significant change in the other circulating biomarkers. ECV did not change significantly during treatment but lower baseline ECV correlated with a greater fall in LVEF. Microscopic changes were seen in the rat myocardium after 6 doses of doxorubicin but electron-microscopy revealed mitochondrial damage after just one dose. Conclusions This translational approach enabled forward and back translation leading to the development of a clinically relevant pre-clinical cardiotoxicity model. Troponin I was the most informative circulating biomarker but peaked at the end of therapy too late to modify treatment and prevent LV decline. The model has the potential to be used to identify earlier biomarkers and evaluate cardioprotective strategies. The imaging findings showed that fibrosis cannot be detected on CMR within one year of chemotherapy but generated a new hypothesis that patients with ‘healthier hearts’ may be a greatest risk of drug-induced cardiotoxicity.

Abstract PL04-04: Developing circulating tumor cell derived explant models (CDX)

Serial biopsies of solid tumors can be challenging, not always without risk to the patient and do not capture intratumoral heterogeneity. ‘Liquid Biopsies’ in the form of Circulating Tumor DNA (ctDNA) and Circulating Tumor Cells (CTCs) offer minimally invasive means to stratify patients for therapy and to routinely monitor therapy responses and anticipate emergent therapy resistance. Whilst technically more challenging than ctDNA, CTCs offer a wider range of biomarker application allowing RNA and protein measurements within single and pools of CTCs following enrichment from the blood. In addition, we have focussed on the development of CTC derived explant models (CDX), particularly in small cell lung cancer (SCLC), where biopsies are particularly difficult to obtain (and often small and necrotic) to study the biology of this aggressive disease and to test new therapies. We have generated 17 SCLC CDX models to date where CTCs, enriched using negative depletion of blood cells, are grown subcutaneously in immune-compromised mice. The take rate is ∼45% and is broadly correlated to the number of CellSearch detected EpCam positive CTCs measured in a paired blood sample. CDX models are derived from SCLC patients at presentation before chemotherapy, where most patients are initially chemosensitive but ∼20% are chemorefractory. For some patients, a matched CDX has been derived following chemotherapy response and subsequent relapse with progressive disease and where a biopsy is rarely acquired. CDX histopathology mimics the diagnostic biopsy of the donor patient and recapitulates the donor patient9s response to chemotherapy. CDX can be passaged with maintained growth characteristics and as few as 5-10 CDX cells can re-establish the tumor consistent with a high frequency of tumor initiating cells. We are exploiting CDX models to explore the biology of SCLC, mechanisms of tumor cell dissemination and chemotherapy resistance. The CDX models are also being used to facilitate drug development. Therapies that show promise in CDX models can be rapidly translated to the clinic where the high prevalence of CTCs in SCLC patients provide minimally invasive, proof of mechanism pharmacodynamic biomarkers and CTC number as a surrogate of tumor response. Once passaged in vivo, tumor cells from disaggregated CDX can be cultured ex vivo over 2-3 weeks for initial and more rapid drug screening. SCLC CDX are also being used to assess the importance of vascular mimicry (VM) - a manifestation of tumor cell plasticity that endows endothelial cell behaviour, and we have shown that VE-Cadherin plays a functional role in SCLC VM, altering tumor growth kinetics and intratumor delivery of cisplatin. The generation of s.c CDX from NSCLC patients is proving more challenging. We have generated a CDX from a NSCLC patient with no CellSearch EpCam positive CTCs in their 7.5ml blood sample but whose blood did contain >150 CTCs/ml blood detected by filtration using an immunofluorescence assay for Epithelial to Mesenchymal Transition. The resultant CDX and the majority of this patient9s CTCs were mesenchymal. Alternative approaches including intrathoracic implantation of enriched NSCLC CTCs are being explored. Subcutaneous CDX have been successfully derived from advanced melanoma patients and can now complement other approaches and extend studies where metastatic melanoma biopsies are unforthcoming. In summary, the development of CDX models yields new opportunities to study advanced disease and examine treatment response and resistance. We are currently developing approaches that will allow the genetic manipulation of CDX cultures to underpin mechanistic studies and we are exploring the generation of CDX across other tumor types. Citation Format: Caroline Dive, Kris Frese, Melanie Galvin, Alice Lallo, Cassandra Hodgkinson, Christopher Morrow, Kathryn Simpson, Francesca Trappani, Stuart Williamson, Lynsey Priest, Romina Girotti, Crispin Miller, Ged Brady, Richard Marais, Fiona Blackhall. Developing circulating tumor cell derived explant models (CDX). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr PL04-04.