Molecular Basis of Microtubule Drug Resistance.
Drugs targeting microtubules are the most effective classes of anticancer agents. However, the emergence of drug resistance in tumor cells has limited their ability to cure disease. Especially in the case of Taxol, most described mechanisms of resistance involve alterations in tubulin. Our lab was the first to describe the presence of tubulin mutations in Taxol-resistant cell lines and this mechanism has been tested in patients.
Androgen Receptor Signaling in Prostate Cancer.
Our long-term goal is to identify novel therapeutic strategies to treat lethal castration-resistant prostate cancer (CRPC), especially those who are resistant to AR pathway inhibitor treatment, and express the AR splice variant, AR-V7. Expression of AR-V7 occurs in approximately 60% of CRPC patients and confers resistance to next-generation AR inhibitors such as enzalutamide and abiraterone as well as to taxanes. Currently there are no other viable therapeutic options for CRPC patients, hence, we are working on developing small molecules that specifically inhibit the expression and the nuclear translocation of AR-V7. These lead small molecules were identified in a rigorous high-throughput small molecule screen of ~170,000 compounds, which identified 13 lead compounds.
We have preliminary data in the lab testing lead compounds by live cell time lapse imaging and fixed cell immunofluorescence using confocal microscopy. We are currently focusing on gaining a mechanistic understanding of how these compounds work and testing them in relevant in vitro and in vivo xenograft models. We will also determine the ability of lead compounds to reverse ASI- and taxane-resistance in vitro and in vivo and develop PROTACS-AR-V7 degrader targeting for the treatment of CRPC.
Liquid Biopsies for Precision Oncology
Metastatic castration resistant prostate cancer and lung cancer are currently incurable, due to treatment resistance. Elucidation of resistance mechanisms requires frequent tumor sampling to monitor tumor evolution and tailor treatments to the individual. Circulating tumor cells (CTCs) represent a non-invasive, accessible “liquid biopsy” source of tumor cells, allowing for longitudinal molecular disease profiling. Circulating tumor cells (CTCs) have successfully been evaluated as a source of tumor material, which can be collected safely and repeatedly. Importantly, the CTC population comprise cells leaving the primary tumor and the several distant metastatic sites, thus recapitulating not only the molecular features of the primary lesion but providing a more comprehensive picture of the disease as a whole. Several strategies to isolate CTCs have been reported in the literature, each based on physical or biological properties specific for this cell population.
Our lab has developed extensive expertise in benchmarking and clinically validating innovative approaches for the isolation of CTCs from the peripheral blood of patients with solid tumors (prostate, gastric, lung and pancreatic cancers). These novel methodologies, based either on positive selection of antigen-expressing CTCs (PSMA-GEDI microfluidic device) or on negative depletion, allowed us to dissect the molecular characteristics of CTCs by targeted (ARv-ddPCR) and untargeted (RNA-seq) transcriptomic profiling and by multiplex confocal microscopy. This molecular interrogation of tumor cells represents the first step to identify the mechanisms underlying tumor resistance to anti-cancer therapies.
Transcriptome Analysis of CTCs
Analysis of RNA-Seq data from Circulating Tumor Cells (CTCs) presents a lot of interesting challenges.Our work centers on developing pipelines and building tools to interpret data across patients, stages and timepoints. Ultimately, we would like to find ways in which we can identify tumor types via expression data to suggest less invasive diagnostic strategies, as well as identify new potential targets. In our most recent work as part of a multi-institutional clinical trial enrolling metastatic castrate resistant prostate cancer (CRPC)- patients treated with the AR signaling inhibitors (ARSi), Abiraterone or Enzalutamide, we performed transcriptome analysis of circulating tumor cells (CTCs) and peripheral blood mononuclear cells (PBMCs) before and after treatment.
CTC RNA-sequencing identified that RB loss and enhanced E2F signaling (as shown in the adjacent figure) along with BRCA loss transcriptional networks were associated with intrinsic ARSi resistance , while inflammatory response signature was significantly associated with acquired resistance. Also, transcriptomic profiling of the circulating immune macroenvironment identified activation of innate immunity with concurrent downregulation of adaptive immunity at progression. These data represent one of the most comprehensive transcriptome analyses using liquid biopsies to identify the molecular underpinnings of clinical drug resistance, providing unique opportunity for therapy optimization in patients with mCRPC.
A CLIP170 Variant Mediates Taxane Resistance
Taxanes, microtubule (MT) stabilizing drugs, are one of the most effective cancer chemotherapeutics with activity against a broad range of tumor types, including breast, prostate, and gastric cancer. Despite their approved use in both first- and second-line therapy, patients commonly exhibit intrinsic resistance. Different resistance mechanisms have been previously described such as tubulin mutations, microtubule post-translational modifications or efflux pumps overexpression, however, none of these alterations have clinical impact.
In our search for mechanisms involved in taxane resistance, we have recently identified a novel variant of the microtubule plus-end binding protein CLIP-170 (we named CLIP-170S) that is associated with taxane resistance by impairing taxanes to bind their target, the microtubules (Thakkar, Kita et al. Dev Cell 2021).
We found that CLIP-170S is expressed in ~60% of gastric cancer tumors and significantly enriched in patients’ refractory to taxane chemotherapy. Importantly, deception of CLIP-170S restores taxane sensitiviy. To identify treatments to overcome CLIP-170S mediated taxane resistance, we employed a computational approach in conjunction with the connectivity map and identified imatinib as a top drug to reverse taxane resistance. We experimentally validated this predicted model showing that imatinib and other RTK inhibitors sensitize taxane-refractory gastric cancer cell lines by selective depletion of CLIP-170S. Our work establishes CLIP-170S as a novel clinically relevant mediator of taxane resistance in GC and the use of RTKi treatment as a viable therapeutic strategy to overcome this resistance. We are currently expanding our research to the potential role of CLIP-170S and other variants of CLIP-170 in taxane-resistance in other different tumor types including prostate, breast and lung.
Drug Target Engagement in Tumor Biopsies from patients
Despite widespread use of taxanes, mechanisms of action and resistance in vivo remain to be established, and there is no way of predicting who will respond to therapy. In this study, we examined prostate cancer (PCa) xenografts and patient samples to identify in vivo mechanisms of taxane action and resistance. Docetaxel drug-target engagement was assessed by confocal anti-tubulin immunofluorescence to quantify microtubule bundling in interphase cells and aberrant mitoses. Tumor biopsies from metastatic PCa patients obtained after their first dose of docetaxel or cabazitaxel were processed to assess microtubule bundling, which correlated with clinical response. Our findings indicate that taxanes target both mitotic and interphase cells in vivo and that resistance is through mechanisms that impair drug-target engagement. Moreover, the findings suggest that microtubule bundling after initial taxane treatment may be a predictive biomarker for clinical response.