CAR affinity modulates the sensitivity of CAR-T cells to PD-1/PD-L1-mediated inhibition.

Chimeric antigen receptor (CAR)-T cell therapy for solid tumors faces significant hurdles, including T-cell inhibition mediated by the PD-1/PD-L1 axis. The effects of disrupting this pathway on T-cells are being actively explored and controversial outcomes have been reported. Here, we hypothesize that CAR-antigen affinity may be a key factor modulating T-cell susceptibility towards the PD-1/PD-L1 axis. We systematically interrogate CAR-T cells targeting HER2 with either low (LA) or high affinity (HA) in various preclinical models. Our results reveal an increased sensitivity of LA CAR-T cells to PD-L1-mediated inhibition when compared to their HA counterparts by using in vitro models of tumor cell lines and supported lipid bilayers modified to display varying PD-L1 densities. CRISPR/Cas9-mediated knockout (KO) of PD-1 enhances LA CAR-T cell cytokine secretion and polyfunctionality in vitro and antitumor effect in vivo and results in the downregulation of gene signatures related to T-cell exhaustion. By contrast, HA CAR-T cell features remain unaffected following PD-1 KO. This behavior holds true for CD28 and ICOS but not 4-1BB co-stimulated CAR-T cells, which are less sensitive to PD-L1 inhibition albeit targeting the antigen with LA. Our findings may inform CAR-T therapies involving disruption of PD-1/PD-L1 pathway tailored in particular for effective treatment of solid tumors.

Comprehensive establishment and characterization of orthoxenograft mouse models of malignant peripheral nerve sheath tumors for personalized medicine.

Malignant peripheral nerve sheath tumors (MPNSTs) are soft-tissue sarcomas that can arise either sporadically or in association with neurofibromatosis type 1 (NF1). These aggressive malignancies confer poor survival, with no effective therapy available. We present the generation and characterization of five distinct MPNST orthoxenograft models for preclinical testing and personalized medicine. Four of the models are patient-derived tumor xenografts (PDTX), two independent MPNSTs from the same NF1 patient and two from different sporadic patients. The fifth model is an orthoxenograft derived from an NF1-related MPNST cell line. All MPNST orthoxenografts were generated by tumor implantation, or cell line injection, next to the sciatic nerve of nude mice, and were perpetuated by 7-10 mouse-to-mouse passages. The models reliably recapitulate the histopathological properties of their parental primary tumors. They also mimic distal dissemination properties in mice. Human stroma was rapidly lost after MPNST engraftment and replaced by murine stroma, which facilitated genomic tumor characterization. Compatible with an origin in a catastrophic event and subsequent genome stabilization, MPNST contained highly altered genomes that remained remarkably stable in orthoxenograft establishment and along passages. Mutational frequency and type of somatic point mutations were highly variable among the different MPNSTs modeled, but very consistent when comparing primary tumors with matched orthoxenografts generated. Unsupervised cluster analysis and principal component analysis (PCA) using an MPNST expression signature of ~1,000 genes grouped together all primary tumor-orthoxenograft pairs. Our work points to differences in the engraftment process of primary tumors compared with the engraftment of established cell lines. Following standardization and extensive characterization and validation, the orthoxenograft models were used for initial preclinical drug testing. Sorafenib (a BRAF inhibitor), in combination with doxorubicin or rapamycin, was found to be the most effective treatment for reducing MPNST growth. The development of genomically well-characterized preclinical models for MPNST allowed the evaluation of novel therapeutic strategies for personalized medicine.

Tubers from patients with tuberous sclerosis complex are characterized by changes in microtubule biology through ROCK2 signalling.

Most patients with tuberous sclerosis complex (TSC) develop cortical tubers that cause severe neurological disabilities. It has been suggested that defects in neuronal differentiation and/or migration underlie the appearance of tubers. However, the precise molecular alterations remain largely unknown. Here, by combining cytological and immunohistochemical analyses of tubers from nine TSC patients (four of them diagnosed with TSC2 germline mutations), we show that alteration of microtubule biology through ROCK2 signalling contributes to TSC neuropathology. All tubers showed a larger number of binucleated neurons than expected relative to control cortex. An excess of normal and altered cytokinetic figures was also commonly observed. Analysis of centrosomal markers suggested increased microtubule nucleation capacity, which was supported by the analysis of an expression dataset from cortical tubers and control cortex, and subsequently linked to under-expression of Rho-associated coiled-coil containing kinase 2 (ROCK2). Thus, augmented microtubule nucleation capacity was observed in mouse embryonic fibroblasts and human fibroblasts deficient in the Tsc2/TSC2 gene product, tuberin. Consistent with ROCK2 under-expression, microtubule acetylation was found to be increased with tuberin deficiency; this alteration was abrogated by rapamycin treatment and mimicked by HDAC6 inhibition. Together, the results of this study support the hypothesis that loss of TSC2 expression can alter microtubule organization and dynamics, which, in turn, deregulate cell division and potentially impair neuronal differentiation.

Genomic imbalance of HMMR/RHAMM regulates the sensitivity and response of malignant peripheral nerve sheath tumour cells to aurora kinase inhibition.

Malignant peripheral nerve sheath tumours (MPNST) are rare, hereditary cancers associated with neurofibromatosis type I. MPNSTs lack effective treatment options as they often resist chemotherapies and have high rates of disease recurrence. Aurora kinase A (AURKA) is an emerging target in cancer and an aurora kinase inhibitor (AKI), termed MLN8237, shows promise against MPNST cell lines in vitro and in vivo. Here, we test MLN8237 against two primary human MPNST grown in vivo as xenotransplants and find that treatment results in tumour cells exiting the cell cycle and undergoing endoreduplication, which cumulates in stabilized disease. Targeted therapies can often fail in the clinic due to insufficient knowledge about factors that determine tumour susceptibilities, so we turned to three MPNST cell-lines to further study and modulate the cellular responses to AKI. We find that the sensitivity of cell-lines with amplification of AURKA depends upon the activity of the kinase, which correlates with the expression of the regulatory gene products TPX2 and HMMR/RHAMM. Silencing of HMMR/RHAMM, but not TPX2, augments AURKA activity and sensitizes MPNST cells to AKI. Furthermore, we find that AURKA activity is critical to the propagation and self-renewal of sphere-enriched MPNST cancer stem-like cells. AKI treatment significantly reduces the formation of spheroids, attenuates the self-renewal of spheroid forming cells, and promotes their differentiation. Moreover, silencing of HMMR/RHAMM is sufficient to endow MPNST cells with an ability to form and maintain sphere culture. Collectively, our data indicate that AURKA is a rationale therapeutic target for MPNST and tumour cell responses to AKI, which include differentiation, are modulated by the abundance of HMMR/RHAMM.

Assessing the RNA effect of 26 DNA variants in the BRCA1 and BRCA2 genes.

Comprehensive genetic testing of the breast cancer susceptibility genes BRCA1 and BRCA2 identified approximately 16% of variants of unknown significance (VUS), a significant proportion of which could affect the correct splicing of the genes. Our aim is to establish a workflow for classifying VUS in these complex genes, the first stage of which is splicing analysis. We used a combined approach consisting of five in silico splicing prediction programs and RT-PCR analysis for a set of 26 variants not previously studied at the mRNA level and six variants that had already been studied, four of which were used as positive controls as they were found to affect the splicing of these genes and the other two were used as negative controls. We identified a splicing defect in 8 of the 26 newly studied variants and ruled out splicing alteration in the remaining 18 variants. The results for the four positive and the two negative control variants were consistent with results presented in the literature. Our results strongly suggest that the combination of RNA analysis and in silico programs is an important step towards the classification of VUS. The results revealed a very high correlation between experimental data and in silico programs when using tools for predicting acceptor/donor sites but a lower correlation in the case of tools for identifying ESE elements.

A mild neurofibromatosis type 1 phenotype produced by the combination of the benign nature of a leaky NF1-splice mutation and the presence of a complex mosaicism.

Here we analyze the genetic and molecular basis responsible for a very benign phenotype observed in an NF1 patient. Quantification of cells carrying the NF1 mutation in different samples derived from the three embryonic layers revealed mosaicism. Furthermore, the construction of a minigene with patient's mutation (c.3198 - 314G>A) confirmed its benign nature due to the leakiness of the splicing mechanism that generated a proportion of correctly spliced transcripts. Hence, we concluded that the mild phenotype observed in this patient is the result of the presence of mosaicism together with the benign nature of a leaky NF1-splice mutation. Finally, with the aim of developing a personalized therapeutic approach for this patient, we demonstrated correction of the splicing defect by using specific antisense morpholino oligomers. Our results provide an example of the molecular complexity behind disease phenotypes and highlight the importance of using comprehensive genetic approaches to better assess phenotype-genotype correlations.