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T cells made with messenger RNA (mRNA) encoding chimeric antigen receptor (CAR) offer a safe alternative to those transduced with viral CARs by mitigating the side effects of constitutively active T cells. Previous studies have shown that mRNA CAR T cells are transiently effective but lack persistence and potency across tumor types. It was hypothesized that the efficacy of mRNA CARs could be improved by utilizing recent advancements in RNA technology, such as incorporating a modified nucleoside, 1-methylpseudouridine, into the mRNA and applying a novel purification method using RNase III to eliminate dsRNA contaminants. T cells electroporated with nucleoside-modified and purified mRNA encoding CD19 CAR showed an initial twofold increase in CAR surface expression, as well as a twofold improvement in cytotoxic killing of leukemia cells that persisted up to 5 days. T cells generated with nucleoside-modified and purified CAR mRNA also showed reduced expression of checkpoint regulators and a differential pattern of genetic activation compared to those made with conventional mRNA. In vivo studies using a leukemia mouse model revealed that the most robust 100-fold suppression of leukemic burden was achieved using T cells electroporated with purified mRNAs, regardless of their nucleoside modification. The results provide a novel approach to generate mRNA for clinical trials, and poise mRNA CAR T cells for increased efficacy during testing as new CAR targets emerge.

Astrocytomas are the most common type of brain tumors in children. Activated BRAF protein kinase mutations are characteristic of pediatric astrocytomas with KIAA1549-BRAF fusion genes typifying low-grade astrocytomas and V600E BRAF alterations characterizing distinct or higher-grade tumors. Recently, BRAF-targeted therapies, such as vemurafenib, have shown great promise in treating V600E-dependent melanomas. Like V600E BRAF, BRAF fusion kinases activate MAPK signaling and are sufficient for malignant transformation; however, here we characterized the distinct mechanisms of action of KIAA1549-BRAF and its differential responsiveness to PLX4720, a first-generation BRAF inhibitor and research analog of vemurafenib. We found that in cells expressing KIAA1549-BRAF, the fusion kinase functions as a homodimer that is resistant to PLX4720 and accordingly is associated with CRAF-independent paradoxical activation of MAPK signaling. Mutagenesis studies demonstrated that KIAA1549-BRAF fusion-mediated signaling is diminished with disruption of the BRAF kinase dimer interface. In addition, the KIAA1549-BRAF fusion displays increased binding affinity to kinase suppressor of RAS (KSR), an RAF relative recently demonstrated to facilitate MEK phosphorylation by BRAF. Despite its resistance to PLX4720, the KIAA1549-BRAF fusion is responsive to a second-generation selective BRAF inhibitor that, unlike vemurafenib, does not induce activation of wild-type BRAF. Our data support the development of targeted treatment paradigms for BRAF-altered pediatric astrocytomas and also demonstrate that therapies must be tailored to the specific mutational context and distinct mechanisms of action of the mutant kinase.

Angiocentric gliomas are pediatric low-grade gliomas (PLGGs) without known recurrent genetic drivers. We performed genomic analysis of new and published data from 249 PLGGs, including 19 angiocentric gliomas. We identified MYB-QKI fusions as a specific and single candidate driver event in angiocentric gliomas. In vitro and in vivo functional studies show that MYB-QKI rearrangements promote tumorigenesis through three mechanisms: MYB activation by truncation, enhancer translocation driving aberrant MYB-QKI expression and hemizygous loss of the tumor suppressor QKI. To our knowledge, this represents the first example of a single driver rearrangement simultaneously transforming cells via three genetic and epigenetic mechanisms in a tumor.

INTRODUCTION: Activating BRAF-fusion mutations occur frequently in pediatric low-grade gliomas (PLGGs). We have previously shown targeting of KIAA1549-BRAF fusion by PLX4720, the research analog of vemurafenib, results in paradoxical activation of the mitogen activated protein kinase (MAPK) signal cascade. We hypothesized that BRAF-fusions could be targeted by downstream MEK inhibitors (MEKi), and that combinatorial targeting of BRAF-fusions could also prevent acquired resistance to single agent therapy. METHODS: BRAF-fusions were screened with a selection of clinically relevant MEKi and with PLX8394, a second-generation RAF inhibitor, in vitro in cell culture and soft agar. BRAF V600E and RAS mutant cells were also screened for comparison. MEKi/PLX8394 resistant BRAF-fusion cell lines were generated in vitro and interrogated by sequencing, immunoblotting, and RNA-seq analysis to identify potential co-targets. Combinatorial therapy was then tested in vitro and in vivo to confirm efficacy. RESULTS: BRAF-fusions in PLGGs can be targeted by MEKi and by the pan-RAF inhibitor PLX8394. AZD6244, MEK162, GDC0623, and GDC0973 all inhibited downstream ERK phosphorylation; however, GSK1120212 more robustly inhibited MAPK activation in the BRAF-fusion, BRAF V600E, and RAS mutant cells. Interestingly, the MEKi/PLX8394 resistant BRAF-fusion cells all showed activation of phosphatidylinositol 3-kinase (PI3K) pathway. BRAF-fusion parental cells were screened with RAD001, a targeted mTOR inhibitor, resulting in modest downstream inhibition. Co-targeting of the parental BRAF-fusions with GSK1120212 and RAD001 resulted in robust inhibition of down-stream signaling and in significant tumor inhibition, and prevented acquired resistance to targeted inhibition. CONCLUSION: Our work demonstrates that BRAF-fusion oncogenes can be effectively targeted with GSK1120212 as single agent but even more robustly with GSK1120212 and RAD001 co-targeting. This provides preclinical rationale for treating PLGGs harboring BRAF-fusion oncogenes with combinatorial MEK and mTOR inhibitors in clinical trial.

Chimeric Antigen Receptor (CAR) T-cells made with mRNA offer a transient and safe alternative to viral CARs, mitigating the concern for persistent unwanted side effects from constitutively active T-cells. Previous studies have shown that mRNA CARs are transiently effective, but lack CAR persistence and have struggled to show success across tumor types. We hypothesized the efficacy of mRNA CARs could be improved by utilizing recent advancements in RNA technology including the use of modified uracil and a novel purification method with RNaseIII to prevent double stranding that induces toxicity. Using the established CD19 CAR model in B-cell acute lymphoblastic leukemia, we created mRNA CARs using previously described methods and compared them to mRNA CARs created using modification of uracil and/or purification to remove any aberrant double stranded mRNA. Comparing the modified and purified mRNA CAR T cells to those created using prior methods of making mRNA, both modified and purified mRNA CAR T cells showed a two-fold increase in expression of the CAR on their surface initially, as well as a two-fold improvement in cytotoxic killing of leukemia cells in vitro that persisted for up to five days. Both the modified and purified mRNA CAR T cells also showed reduced expression of negative checkpoint regulators compared to original RNA CAR T cells. However, in vivo studies using a patient-derived xenograft model with a single dose of CAR T cells revealed purified RNA CAR T cells offered the most robust 2-log enhanced suppression of leukemic burden. RNaseIII is a novel purification technique that has not yet been reported in the literature for RNA manufacturing. Our results provide a time efficient purification method that can be easily incorporated into RNA production for use in clinical trials, and poise RNA CARs for increased efficacy as new CAR targets emerge and are being tested. Additional studies are ongoing investigating if the observed improved efficacy will translate to improved cytotoxicity in solid tumor models. Citation Format: Jessica Foster, Namrata Choudhari, Adam Resnick, David Barrett. Novel mRNA purification method with RNaseIII improves efficacy of RNA chimeric antigen receptor T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5614. doi:10.1158/1538-7445.AM2017-5614

Chimeric Antigen Receptor (CAR) T-cells made with mRNA offer a transient and safe alternative to viral CARs, mitigating the concern for persistent unwanted side effects from constitutively active T-cells. Previous studies have shown that mRNA CARs are transiently effective, but lack CAR persistence and have struggled to show success across tumor types. We hypothesized the efficacy of mRNA CARs could be improved by utilizing recent advancements in RNA technology including the use of modified nucleosides and novel purification methods of the RNA to prevent double stranding that induces toxicity. Using the established CD19 CAR model in B-cell leukemia, we created mRNA CARs using previously described methods and compared them to mRNA CARs created using the modifications of pseudo-uridine or N-methyl pseudo-uridine instead of uridine, and purification to remove any aberrant double stranded mRNA. Comparing the modified and purified mRNA CAR T cells to those created using prior methods of making mRNA, the novel mRNA CAR T cells showed a two-fold increase in expression of the CAR on their surface initially, as well as a two-fold improvement in cytotoxic killing of leukemia cells that persisted for up to five days. The modified and purified mRNA CAR T cells also showed reduced expression of negative checkpoint regulators compared to original RNA CAR T cells. In vivo studies using a leukemia mouse model with a single dose of CAR T cells also showed marked improvement using modified and purified mRNA CAR T cells, with purified RNA showing the most robust 2-log enhanced suppression of leukemic burden. Our results provide a novel technique that can be easily incorporated into RNA production for use in clinical trials, and poise RNA CARs for increased efficacy as new CAR targets emerge and are being tested. Citation Format: Jessica Foster, Namrata Choudhari, David Barrett, Adam Resnick. Novel mRNA manufacturing techniques using modification of nucleosides and purification improves the efficacy of mRNA chimeric antigen receptor T cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B083.

The second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ), formed by the p110 family of PI3-kinases, promotes cellular growth, proliferation, and survival, in large part by activating the protein kinase Akt/PKB. We show that inositol polyphosphate multikinase (IPMK) physiologically generates PIP 3 as well as water soluble inositol phosphates. IPMK deletion reduces growth factor-elicited Akt signaling and cell proliferation caused uniquely by loss of its PI3-kinase activity. Inhibition of p110 PI3-kinases by wortmannin prevents IPMK phosphorylation and activation. Thus, growth factor stimulation of Akt signaling involves PIP 3 generation through the sequential activations of the p110 PI3-kinases and IPMK. As inositol phosphates inhibit Akt signaling, IPMK appears to act as a molecular switch, inhibiting or stimulating Akt via its inositol phosphate kinase or PI3-kinase activities, respectively. Drugs regulating IPMK may have therapeutic relevance in influencing cell proliferation.

Low-grade gliomas are the most common tumors of the central nervous system in children. We recently described a 7q34 duplication resulting in a novel KIAA1549-BRAF fusion, that includes the C-terminus BRAF kinase domain, exclusively in the majority of pediatric low-grade gliomas. BRAF activating mutations have been well described in human malignancies; however, little is known about KIAA1549. In silico analysis of the fusion gene predicts two putative transmembrane domains in the N-terminus segment of KIAA1549. Clones of the fusion were created, including the long and short forms found in pediatric tumors and a truncated version lacking a transmembrane domain. NIH/3T3 stable cell lines for each clone were then generated using Plat-E retrovirus. Assays were also performed with transient over-expression of the clones in HEK/293T cells. Activity of the fusion clones was compared to BRAF (wild-type, kinase-dead, V600E mutant), KIAA1549 (wild-type), and Rasv12 mutant. The KIAA1549-BRAF fusion gene constructs all demonstrated activation of the MAPK pathway with increased phosphylation of both MEK 1/2 and ERK1/2; malignant transformation in soft agar assay; and increased transcriptional output. In vivo studies are ongoing but preliminary results demonstrate the KIAA1549-BRAF fusion is sufficient to cause tumor formation in a xenograft murine model. Overall, our sets of experiments reveal the truncated fusion has kinase activity comparable to the canonical V600E BRAF mutant. Wild-type KIAA1549 had no effect on MEK1/2 and ERK1/2 phosphorylation nor does over-expression cause cell transformation. Not surprising RAF specific and MEK specific inhibition resulted in decreased MEK1/2 phosphorylation in BRAF (all variants) and KIAA1549-BRAF fusion transient over-expression in HEK/293T cells and in IP/BRAF kinase assays. Interestingly, RAF specific inhibition of the KIAA1549-BRAF fusion NIH/3T3 stable cell lines resulted in increased MEK 1/2 phosphorylation at low drug concentrations (0.01-10 micromolar) consistent with recent reports describing paradoxical activation of the MAPK pathway in wild-type BRAF. Drug concentrations of greater than 20 micromolar demonstrated inhibition of all of the constructs. Our data suggest the KIAA1549-BRAF fusion gene has oncogenic potential and is sufficient for tumor formation; however, BRAF kinase specific inhibition can result in paradoxical activation of the MAPK pathway at lower drug concentrations. Targeting the KIAA1549-BRAF fusion in pediatric low-grade gliomas is likely still a rational approach but further preclinical studies are needed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3457. doi:10.1158/1538-7445.AM2011-3457