Tuning microtubule dynamics to enhance cancer therapy by modulating FER-mediated CRMP2 phosphorylation

Though used widely in cancer therapy, paclitaxel only elicits a response in a fraction of patients. A strong determinant of paclitaxel tumor response is the state of microtubule dynamic instability. However, whether the manipulation of this physiological process can be controlled to enhance paclitaxel response has not been tested. Here, we show a previously unrecognized role of the microtubule-associated protein CRMP2 in inducing microtubule bundling through its carboxy terminus. This activity is significantly decreased when the FER tyrosine kinase phosphorylates CRMP2 at Y479 and Y499. The crystal structures of wild-type CRMP2 and CRMP2-Y479E reveal how mimicking phosphorylation prevents tetramerization of CRMP2. Depletion of FER or reducing its catalytic activity using sub-therapeutic doses of inhibitors increases paclitaxel-induced microtubule stability and cytotoxicity in ovarian cancer cells and in vivo. This work provides a rationale for inhibiting FER-mediated CRMP2 phosphorylation to enhance paclitaxel on-target activity for cancer therapy

On the origin, progression, and evolution of ovarian cancer

High-grade serous ovarian cancer (HGSOC) is amongst the deadliest of cancers. Despite decades of research on this disease, the exact mechanisms through which this carcinoma initiates, spreads, and relapses remain unclear. I believe the main reason behind this discrepancy between importance and available knowledge roots in the fact that currently available genomics methods are not sensitive enough for accurate study of limited amount of clinical materials. To address this issue, I optimised a novel library preparation protocol called DigiPico to generate reliable next generation sequencing data from picogram quantities of DNA that can be obtained from 10-20 human cells. Moreover, I implemented DigiTitan and MutLX analysis algorithms to identify clone-specific copy number alterations and small nucleotide variations from DigiPico data. Validating MutLX algorithm using experimental and simulated data I demonstrated that DigiPico/MutLX is a uniquely sensitive, accurate, and reliable method for analysis of micro-heterogeneity in solid tumours. Next, using DigiPico, I conducted the first accurate case study of genetic micro-heterogeneity on a collection of pre-chemotherapy tumour islets and post-chemotherapy microscopic residual disease (MRD) samples from one HGSOC patient. Analysing the results using DigiTitan and MutLX, firstly, I introduced tumour heterogeneity and genetic diversity as a hallmark of HGSOC. Secondly, based on new observations from this study, I challenged a pre-existing notion that mutations in TP53 gene are responsible for the initiation of HGSOC. I proposed that mutations in the regulatory regions that adversely affect the DNA repair machinery which can lead to subsequent large-scale genomic alterations precede the loss of TP53 function in the tumorigenesis pathway of HGSOC. Thirdly, comparing the somatic mutation load of pre-chemotherapy, MRD and recurrent tumours, I demonstrated that MRD harbours the least number of somatic mutations. Thereby, I hypothesized that MRD is likely to be composed of stem cell-like tumour cells that have survived the chemotherapy therefore suggesting that MRD is likely be the recurrence precursor. Lastly, I propose experimental approaches that are required to effectively validate or nullify these hypotheses.</p

On the origin, progression, and evolution of ovarian cancer

High-grade serous ovarian cancer (HGSOC) is amongst the deadliest of cancers. Despite decades of research on this disease, the exact mechanisms through which this carcinoma initiates, spreads, and relapses remain unclear. I believe the main reason behind this discrepancy between importance and available knowledge roots in the fact that currently available genomics methods are not sensitive enough for accurate study of limited amount of clinical materials. To address this issue, I optimised a novel library preparation protocol called DigiPico to generate reliable next generation sequencing data from picogram quantities of DNA that can be obtained from 10-20 human cells. Moreover, I implemented DigiTitan and MutLX analysis algorithms to identify clone-specific copy number alterations and small nucleotide variations from DigiPico data. Validating MutLX algorithm using experimental and simulated data I demonstrated that DigiPico/MutLX is a uniquely sensitive, accurate, and reliable method for analysis of micro-heterogeneity in solid tumours. Next, using DigiPico, I conducted the first accurate case study of genetic micro-heterogeneity on a collection of pre-chemotherapy tumour islets and post-chemotherapy microscopic residual disease (MRD) samples from one HGSOC patient. Analysing the results using DigiTitan and MutLX, firstly, I introduced tumour heterogeneity and genetic diversity as a hallmark of HGSOC. Secondly, based on new observations from this study, I challenged a pre-existing notion that mutations in TP53 gene are responsible for the initiation of HGSOC. I proposed that mutations in the regulatory regions that adversely affect the DNA repair machinery which can lead to subsequent large-scale genomic alterations precede the loss of TP53 function in the tumorigenesis pathway of HGSOC. Thirdly, comparing the somatic mutation load of pre-chemotherapy, MRD and recurrent tumours, I demonstrated that MRD harbours the least number of somatic mutations. Thereby, I hypothesized that MRD is likely to be composed of stem cell-like tumour cells that have survived the chemotherapy therefore suggesting that MRD is likely be the recurrence precursor. Lastly, I propose experimental approaches that are required to effectively validate or nullify these hypotheses.</p

Consequences of resistance evolution in a Cas9-based sex conversion-suppression gene drive for insect pest management.

The use of a site-specific homing-based gene drive for insect pest control has long been discussed, but the easy design of such systems has become possible only with the recent establishment of CRISPR/Cas9 technology. In this respect, novel targets for insect pest management are provided by new discoveries regarding sex determination. Here, we present a model for a suppression gene drive designed to cause an all-male population collapse in an agricultural pest insect. To evaluate the molecular details of such a sex conversion-based suppression gene drive experimentally, we implemented this strategy in Drosophila melanogaster to serve as a safe model organism. We generated a Cas9-based homing gene-drive element targeting the transformer gene and showed its high efficiency for sex conversion from females to males. However, nonhomologous end joining increased the rate of mutagenesis at the target site, which resulted in the emergence of drive-resistant alleles and therefore curbed the gene drive. This confirms previous studies that simple homing CRISPR/Cas9 gene-drive designs will be ineffective. Nevertheless, by performing population dynamics simulations using the parameters we obtained in D. melanogaster and by adjusting the model for the agricultural pest Ceratitis capitata, we were able to identify adequate modifications that could be successfully applied for the management of wild Mediterranean fruit fly populations using our proposed sex conversion-based suppression gene-drive strategy

A highly accurate platform for clone-specific mutation discovery enables the study of active mutational processes

Bulk whole genome sequencing (WGS) enables the analysis of tumor evolution but, because of depth limitations, can only identify old mutational events. The discovery of current mutational processes for predicting the tumor's evolutionary trajectory requires dense sequencing of individual clones or single cells. Such studies, however, are inherently problematic because of the discovery of excessive false positive mutations when sequencing picogram quantities of DNA. Data pooling to increase the confidence in the discovered mutations, moves the discovery back in the past to a common ancestor. Here we report a robust whole genome sequencing and analysis pipeline (DigiPico/MutLX) that virtually eliminates all false positive results while retaining an excellent proportion of true positives. Using our method, we identified, for the first time, a hyper-mutation (kataegis) event in a group of ∼30 cancer cells from a recurrent ovarian carcinoma. This was unidentifiable from the bulk WGS data. Overall, we propose DigiPico/MutLX method as a powerful framework for the identification of clone-specific variants at an unprecedented accuracy

Adipocyte-like signature in ovarian cancer minimal residual disease identifies metabolic vulnerabilities of tumor initiating cells

Similar to tumor initiating cells (TICs), minimal residual disease (MRD) is capable of re-initiating tumors and causing recurrence. However, the molecular characteristics of solid tumor MRD cells and drivers of their survival have remained elusive. Here we performed dense multi-region transcriptomics analysis of paired biopsies from 17 ovarian cancer patients before and after chemotherapy. We reveal that while MRD cells share important molecular signatures with TICs, they are also characterized by an adipocyte-like gene expression signature and a portion of them had undergone epithelial-mesenchymal transition (EMT). In a cell culture MRD model, MRD-mimic cells show the same phenotype and are dependent on fatty acid oxidation for survival and resistance to cytotoxic agents. These findings identify EMT and FAO as attractive targets to eradicate MRD in ovarian cancer and make a compelling case for the further testing of FAO inhibitors in treating MRD

Tuning microtubule dynamics to enhance cancer therapy by modulating FER-mediated CRMP2 phosphorylation

Though used widely in cancer therapy, paclitaxel only elicits a response in a fraction of patients. A strong determinant of paclitaxel tumor response is the state of microtubule dynamic instability. However, whether the manipulation of this physiological process can be controlled to enhance paclitaxel response has not been tested. Here, we show a previously unrecognized role of the microtubule-associated protein CRMP2 in inducing microtubule bundling through its carboxy terminus. This activity is significantly decreased when the FER tyrosine kinase phosphorylates CRMP2 at Y479 and Y499. The crystal structures of wild-type CRMP2 and CRMP2-Y479E reveal how mimicking phosphorylation prevents tetramerization of CRMP2. Depletion of FER or reducing its catalytic activity using sub-therapeutic doses of inhibitors increases paclitaxel-induced microtubule stability and cytotoxicity in ovarian cancer cells and in vivo. This work provides a rationale for inhibiting FER-mediated CRMP2 phosphorylation to enhance paclitaxel on-target activity for cancer therapy