Disabling VEGF-response of Purkinje Cells by downregulation of KDR\it KDR via miRNA-204-5p

The vascular endothelial growth factor (VEGF) is well known for its wide-ranging functions, not only in the vascular system, but also in the central (CNS) and peripheral nervous system (PNS). To study the role of VEGF in neuronal protection, growth and maturation processes have recently attracted much interest. These effects are mainly mediated by VEGF receptor 2 (VEGFR-2). Current studies have shown the age-dependent expression of VEGFR-2 in Purkinje cells (PC), promoting dendritogenesis in neonatal, but not in mature stages. We hypothesize that microRNAs (miRNA/miR) might be involved in the regulation of VEGFR-2 expression during the development of PC. In preliminary studies, we performed a miRNA profiling and identified miR204-5p as a potential regulator of VEGFR-2 expression. In the recent study, organotypic slice cultures of rat cerebella (postnatal day (p) 1 and 9) were cultivated and VEGFR-2 expression in PC was verified via immunohistochemistry. Additionally, PC at age p9 and p30 were isolated from cryosections by laser microdissection (LMD) to analyse VEGFR-2 expression by quantitative RT-PCR. To investigate the influence of miR204-5p on VEGFR-2 levels in PC, synthetic constructs including short hairpin (sh)-miR204-5p cassettes (miRNA-mimics), were microinjected into PC. The effects were analysed by confocal laser scanning microscopy (CLSM) and morphometric analysis. For the first time, we could show that miR204-5p has a negative effect on VEGF sensitivity in juvenile PC, resulting in a significant decrease of dendritic growth compared to untreated juvenile PC. In mature PC, the overexpression of miR204-5p leads to a shrinkage of dendrites despite VEGF treatment. The results of this study illustrate, for the first time, which miR204-5p expression has the potential to play a key role in cerebellar development by inhibiting VEGFR-2 expression in PC

Digital-droplet PCR for quantification of CD19-directed CAR T-cells

CD19-directed CAR-T-cells (CD19-CAR) have demonstrated remarkable clinical results in patients suffering from refractory or relapsed lymphoma and acute lymphoblastic leukemia. In order to further optimize follow-up, to explain treatment failure, and to control adverse events biomarkers for monitoring of response are urgently needed. Peak expansion and persistence are correlated with response rates and severity of side effects. However, no standardized method or commercially assay for CD19-CAR measurement is established yet. In this study, two primer-probe assays for digital-droplet PCR (ddPCR) were designed and subsequently explored on 54 samples collected from seven patients after CD19-CAR treatment with axi-cel over time. Detection and quantification of CAR-T-cells were feasible and reliable for all patients included. Peak expansion measured with our assay significantly correlated with the grade of neurologic adverse events but not with cytokine release syndrome. All patients with loss of CAR-signal eventually had disease progression. In summary, our novel assay allows monitoring of CAR-T-cells $\textit {in vivo}$ and may add to safety and efficacy of CAR-T treatment

Altered T-lymphocyte biology following high-dose melphalan and autologous stem cell transplantation with implications for adoptive T-cell therapy

In relapsed and refractory multiple myeloma (MM), adoptive cell therapies (ACT) including CAR-T-cells are under clinical investigation. However, relapse due to T-cell exhaustion or limited persistence is an obstacle. Before ACT are considered in MM, high-dose (HD) melphalan followed by autologous stem-cell transplantation (autoSCT) has been administered in most clinical situations. Yet, the impact of HD chemotherapy on T-cells in MM with respect to ACT is unclear. In this study, T-lymphocytes’ phenotypes, expansion properties, lentiviral transduction efficacy, and gene expression were examined with special respect to patients following HD melphalan. Significant impairment of T-cells’ expansion and transduction rates could be demonstrated. Expansion was diminished due to inherent disadvantages of the predominant T-cell phenotype but restored over time. The quantitative fraction of CD27−/CD28− T-cells before expansion was predictive of T-cell yield. Following autoSCT, the transduction efficacy was reduced by disturbed lentiviral genome integration. Moreover, an unfavorable T-cell phenotype after expansion was demonstrated. In initial analyses of CD107a degranulation impaired T-cell cytotoxicity was detected in one patient following melphalan and autoSCT. The findings of our study have potential implications regarding the time point of leukapheresis for CAR-T-cell manufacturing. Our results point to a preferred interval of more than 3 months until patients should undergo cell separation for CAR-T therapy in the specific situation post-HD melphalan/autoSCT. Monitoring of CD27−/CD28− T-cells, has the potential to influence clinical decision making before apheresis in MM

Secondary resistance to anti-EGFR therapy by transcriptional reprogramming in patient-derived colorectal cancer models

$\bf Background$ The development of secondary resistance (SR) in metastatic colorectal cancer (mCRC) treated with anti-epidermal growth factor receptor (anti-EGFR) antibodies is not fully understood at the molecular level. Here we tested in vivo selection of anti-EGFR SR tumors in CRC patient-derived xenograft (PDX) models as a strategy for a molecular dissection of SR mechanisms. $\bf Methods$ We analyzed 21 $\textit {KRAS, NRAS, BRAF,}$ and $\it PI3K$ wildtype CRC patient-derived xenograft (PDX) models for their anti-EGFR sensitivity. Furthermore, 31 anti-EGFR SR tumors were generated via chronic in vivo treatment with cetuximab. A multi-omics approach was employed to address molecular primary and secondary resistance mechanisms. Gene set enrichment analyses were used to uncover SR pathways. Targeted therapy of SR PDX models was applied to validate selected SR pathways. $\bf Results$ In vivo anti-EGFR SR could be established with high efficiency. Chronic anti-EGFR treatment of CRC PDX tumors induced parallel evolution of multiple resistant lesions with independent molecular SR mechanisms. Mutations in driver genes explained SR development in a subgroup of CRC PDX models, only. Transcriptional reprogramming inducing anti-EGFR SR was discovered as a common mechanism in CRC PDX models frequently leading to RAS signaling pathway activation. We identified cAMP and STAT3 signaling activation, as well as paracrine and autocrine signaling via growth factors as novel anti-EGFR secondary resistance mechanisms. Secondary resistant xenograft tumors could successfully be treated by addressing identified transcriptional changes by tailored targeted therapies. $\bf Conclusions$ Our study demonstrates that SR PDX tumors provide a unique platform to study molecular SR mechanisms and allow testing of multiple treatments for efficient targeting of SR mechanisms, not possible in the patient. Importantly, it suggests that the development of anti-EGFR tolerant cells via transcriptional reprogramming as a cause of anti-EGFR SR in CRC is likely more prevalent than previously anticipated. It emphasizes the need for analyses of SR tumor tissues at a multi-omics level for a comprehensive molecular understanding of anti-EGFR SR in CRC

Lasting response by vertical inhibition with cetuximab and trametinib in KRAS\it KRAS-mutated colorectal cancer patient-derived xenografts

Although approximately half of all metastatic colorectal cancers (mCRCs) harbour mutations in $\it KRAS$ or $\it NRAS$, hardly any progress has been made regarding targeted treatment for this group over the last few years. Here, we investigated the efficacy of vertical inhibition of the RAS-pathway by targeting epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase kinase (MEK) in patient-derived xenograft (PDX) tumours with primary $\it KRAS$ mutation. In total, 19 different PDX models comprising 127 tumours were tested. Responses were evaluated according to baseline tumour volume changes and graded as partial response (PR; ≤ − 30%), stable disease (SD; between −30% and +20%) or progressive disease (PD; ≥ + 20%). Vertical inhibition with trametinib and cetuximab induced SD or PR in 74% of analysed models, compared to 24% by monotherapy with trametinib. In cases of PR by vertical inhibition (47%), responses were lasting (as long as day 137), with a low incidence of secondary resistance (SR). Molecular analyses revealed that primary and SR was driven by transcriptional reprogramming activating the RAS pathway in a substantial fraction of tumours. Together, these preclinical data strongly support the translation of this combination therapy into clinical trials for CRC patients