Despite all currently available anti-pandemic monoclonal-antibodies (mAbs) and vaccines, subsequently emerging pandemic-infections will likely become more pan-resistant-, -transmissible and/or -lethal. We have created HEDGES generation-2, a significantly more-combinatorial, -synergistic version of our generation-1 HEDGES DNA vector-based platform. We previously published that one safe intravenous injection of a HEDGES generation-1 DNA vector encoding one of three different FDA-approved mAbs produced durable therapeutic serum mAb levels as well as critical therapeutic endpoints in immunocompetent mice. Here we show one safe, intravenous administration of a 2nd-generation HEDGES DNA vector co-encoding four different anti-SARS-CoV-2 mAbs rapidly then durably co-produces high anti-SARS-CoV-2 mAb serum levels that effectively block SARS-CoV-2 virus binding to the ACE-2 spike protein in immunocompetent mice. In addition, four weekly intravenous HEDGES generation-2 DNA vector administrations co-encoding a total of ten-different anti-SARS-CoV-2 mAbs, 5J8, plus an anti-1918 pandemic influenza mAb and mepolizumab, an FDA-approved anti-IL-5 mAb, durably co-produce highly-neutralizing 5J8 anti-pandemic influenza mAb serum levels, as well as durably block SARS-CoV-2 virus-ACE-2 receptor binding in mice. Furthermore, unlike vaccines and mAbs, HEDGES does not require an intact cold chain and is readily freeze dried, enabling its prolonged storage at ambient temperatures worldwide, even in equatorial regions. Also, HEDGES can create, then deploy novel, more effective anti-pandemic mAbs ~three weeks after their identification. Conversely, vaccines require ~three months to deploy, recombinant-mAbs ~nine months. By rapidly then durably co-producing many different highly-neutralizing, highly-synergistic anti-pandemic mAbs, HEDGES may effectively co-prevent both SARS-CoV-2 and pandemic-influenza infections. HEDGES may also prevent even more-transmissible, -pan-resistant and/or -lethal pandemic diseases that subsequently-emerge.

Text of Supplementary Figure Legends

Supplementary Table 7 gene_importance_t0.1_limit4_ew

Few effective therapeutic options exist after progression on immune checkpoint blockade (ICB) for melanoma. Here, we utilized a platform incorporating transcriptomic profiling, high-throughput drug screening, and murine models to demonstrate the preclinical efficacy of several combinatorial regimens against ICB-resistant melanoma. Transcriptomic analysis of ICB-resistant melanomas demonstrated activation of several targetable pathways. High-throughput drug screening targeting these pathways identified several effective combinations in ICB-resistant patient-derived xenograft models. The combination of cobimetinib and regorafenib (termed Cobi+Reg) emerged as a particularly promising regimen, with efficacy against distinct molecular melanoma subtypes and after progression on ICB in immunocompetent models. Transcriptomic and spatial analysis of Cobi+Reg-treated tumors demonstrated upregulation of antigen presentation machinery, with concomitantly increased activated T cell infiltration. Combining Cobi+Reg with ICB was superior to either modality in vivo. This analytical platform exploits the biology of ICB-resistant melanoma to identify therapeutic vulnerabilities, resulting in the identification of drug combinations that form the basis for rational clinical trial design in the setting of advanced melanoma resistant to ICB.

Supplementary Table 8 FGFR mutations

Supplementary Table 4 LOBICO PDXC % sens vs insens

Supplementary Table 6 ROC formulae training and test

Supplementary Table 3 binarization sensitivity 19 drugs

Supplementary Table 2 pancancer clinical info

Supplementary Figure 2 from Cannabidiol Enhances the Inhibitory Effects of Δ9-Tetrahydrocannabinol on Human Glioblastoma Cell Proliferation and Survival

Abstract As a result of tumor heterogeneity and solid cancers harboring multiple molecular defects, precision medicine platforms in oncology are most effective when both genetic and pharmacologic determinants of a tumor are evaluated. Expandable patient-derived xenograft (PDX) mouse tumor and corresponding PDX culture (PDXC) models recapitulate many of the biological and genetic characteristics of the original patient tumor, allowing for a comprehensive pharmacogenomic analysis. Here, the somatic mutations of 23 matched patient tumor and PDX samples encompassing four cancers were first evaluated using next-generation sequencing (NGS). 19 antitumor agents were evaluated across 78 patient-derived tumor cultures using clinically relevant drug exposures. A binarization threshold sensitivity classification determined in culture (PDXC) was used to identify tumors that best respond to drug in vivo (PDX). Using this sensitivity classification, logic models of DNA mutations were developed for 19 antitumor agents to predict drug response. We determined that the concordance of somatic mutations across patient and corresponding PDX samples increased as variant allele frequency increased. Notable individual PDXC responses to specific drugs, as well as lineage-specific drug responses were identified. Robust responses identified in PDXC were recapitulated in vivo in PDX-bearing mice and logic modeling determined somatic gene mutation(s) defining response to specific antitumor agents. In conclusion, combining NGS of primary patient tumors, high-throughput drug screen using clinically relevant doses, and logic modeling, can provide a platform for understanding response to therapeutic drugs targeting cancer.

Supplementary Table 1, Figure Legends 1-2 from Cannabidiol Enhances the Inhibitory Effects of Δ9-Tetrahydrocannabinol on Human Glioblastoma Cell Proliferation and Survival

The longitudinal monitoring of patient circulating tumor DNA (ctDNA) provides a powerful method for tracking the progression, remission, and recurrence of several types of cancer. Often, clinical and research approaches involve the manual review of individual liquid biopsy reports after sampling and genomic testing. Here, we describe a process developed to integrate techniques utilized in data science within a cancer research framework. Using data collection, an analysis that classifies genetic cancer mutations as pathogenic, and a patient matching methodology that identifies the same donor within all liquid biopsy reports, the manual work for research personnel is drastically reduced. Automated dashboards provide longitudinal views of patient data for research studies to investigate tumor progression and treatment efficacy via the identification of ctDNA variant allele frequencies over time.

Supplementary Figure 1 from Cannabidiol Enhances the Inhibitory Effects of Δ9-Tetrahydrocannabinol on Human Glioblastoma Cell Proliferation and Survival

Glioblastoma's (GBM) aggressive growth is driven by redundant activation of a myriad of signaling pathways and genomic alterations in tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR), which is altered in over 50% of cases. Single agents targeting EGFR have not proven effective against GBM. In this study, we aimed to identify an effective anti-tumor regimen using pharmacogenomic testing of patient-derived GBM samples, in culture and in vivo. High-throughput pharmacological screens of ten EGFR-driven GBM samples identified the combination of erlotinib (EGFRi) and MLN0128 (a mammalian target of rapamycin inhibitor, or MTORi) as the most effective at inhibiting tumor cell viability. The anti-tumor activity of erlonitib+MLN0128 was synergistic and produced inhibition of the p-EGFR, mitogen-activated protein kinase (MAPK), and Phosphoinositide 3-kinase (PI3K) pathways in culture. Using an orthotopic murine model of GBM, we show that erlotinib+MLN0128 inhibited tumor growth in vivo and significantly prolonged the survival of tumor-bearing mice. Expression profiling of tumor tissues from treated mice revealed a unique gene signature induced by erlotinib+MLN0128, consisting of downregulation of immunosuppressive chemokines in the tumor microenvironment, including C-C motif chemokine ligand 2 (CCL2) and periostin. Lower periostin levels resulted in the inhibition of Iba1+ (tumor-promoting) macrophage infiltration of GBM xenografts. Taken together, our results demonstrate that pharmacological co-targeting of EGFR and MTOR using clinically available drugs represents an effective treatment paradigm for EGFR-driven GBMs, acting both by inhibiting tumor cell growth and modulating the immune tumor microenvironment.

BACKGROUND:Diffuse midline gliomas (DMG) are highly invasive brain tumors with rare survival beyond two years past diagnosis and limited understanding of the mechanism behind tumor invasion. Previous reports demonstrate upregulation of the protein ID1 with H3K27M and ACVR1 mutations in DMG, but this has not been confirmed in human tumors or therapeutically targeted. METHODS:Whole exome, RNA, and ChIP-sequencing was performed on the ID1 locus in DMG tissue. Scratch-assay migration and transwell invasion assays of cultured cells were performed following shRNA-mediated ID1-knockdown. In vitro and in vivo genetic and pharmacologic [cannabidiol (CBD)] inhibition of ID1 on DMG tumor growth was assessed. Patient-reported CBD dosing information was collected. RESULTS:Increased ID1 expression in human DMG and in utero electroporation (IUE) murine tumors is associated with H3K27M mutation and brainstem location. ChIP-sequencing indicates ID1 regulatory regions are epigenetically active in human H3K27M-DMG tumors and prenatal pontine cells. Higher ID1-expressing astrocyte-like DMG cells share a transcriptional program with oligo/astrocyte-precursor cells (OAPCs) from the developing human brain and demonstrate upregulation of the migration regulatory protein SPARCL1. Genetic and pharmacologic (CBD) suppression of ID1 decreases tumor cell invasion/migration and tumor growth in H3.3/H3.1K27M PPK-IUE and human DIPGXIIIP* in vivo models of pHGG. The effect of CBD on cell proliferation appears to be non-ID1 mediated. Finally, we collected patient-reported CBD treatment data, finding that a clinical trial to standardize dosing may be beneficial. CONCLUSIONS:H3K27M-mediated re-activation of ID1 in DMG results in a SPARCL1+ migratory transcriptional program that is therapeutically targetable with CBD.

PDF file - 2804K, Supplementary Figure 1. Id-1 levels are increased in the CD133+ GBM cells. Supplementary Figure 2. Id-1 knockdown inhibits U251 cell neurosphere formation. Supplementary Figure 3. Id-1 knockdown in GBM cells increases survival in an orthotopic mouse model. Supplementary Figure 4. CBD treatment reduces phosphorylation of several kinases involved in cell survival and aggressiveness. Supplementary Figure 5. CBD reduces tumor progression in vivo. Supplementary Table 1. Primary cultures analyzed by immunofluorescence or Western blot.

Therapy of BRAF -mutant melanoma with selective inhibitors of BRAF (BRAFi) and MEK (MEKi) represents a major clinical advance but acquired resistance to therapy has emerged as a key obstacle. To date, no clinical approaches successfully resensitize to BRAF/MEK inhibition. Here, we develop a therapeutic strategy for melanoma using bromosporine, a bromodomain inhibitor. Bromosporine (bromo) monotherapy produced significant anti-tumor effects against established melanoma cell lines and patient-derived xenografts (PDXs). Combinatorial therapy involving bromosporine and cobimetinib (bromo/cobi) showed synergistic anti-tumor effects in multiple BRAFi-resistant PDX models. The bromo/cobi combination was superior in vivo to standard BRAFi/MEKi therapy in the treatment-naive BRAF -mutant setting and to MEKi alone in the setting of immunotherapy-resistant NRAS - and NF1 -mutant melanoma. RNA sequencing of xenografts treated with bromo/cobi revealed profound down-regulation of genes critical to cell division and mitotic progression. Bromo/cobi treatment resulted in marked DNA damage and cell-cycle arrest, resulting in induction of apoptosis. These studies introduce bromodomain inhibition, alone or combined with agents targeting the mitogen activated protein kinase pathway, as a rational therapeutic approach for melanoma refractory to standard targeted or immunotherapeutic approaches.

Background Cannabidiol (CBD), a nonpsychoactive cannabinoid with a low toxicity profile, has been shown to produce antitumor activity across cancers in part through selective production of reactive oxygen species (ROS) in tumor cells. The alkylating agent, temozolomide (TMZ), is standard of care for treatment of glioblastoma (GBM). It can trigger increased ROS to induce DNA damage. It has also been reported that downregulating the expression of RAD51, an important DNA damage repair protein, leads to sensitization of GBM to TMZ. Methods We determined the extent to which CBD enhanced the antitumor activity of TMZ in multiple orthotopic models of GBM. In addition, we investigated the potential for CBD to enhance the antitumor activity of TMZ through production of ROS and modulation of DNA repair pathways. Results CBD enhanced the activity of TMZ in U87 MG and U251 GBM cell lines and in patient-derived primary GBM163 cells leading to stimulation of ROS, activation of the ROS sensor AMP-activated protein kinase (AMPK), and upregulation of the autophagy marker LC3A. CBD produced a sensitization of U87 and GBM163-derived intracranial (i.c.) tumors to TMZ and significantly increased survival of tumor-bearing mice. However, these effects were not observed in orthotopic models derived from GBM with intact methylguanine methyltransferase (MGMT) expression. We further demonstrate that CBD inhibited RAD51 expression in MGMT-methylated models of GBM, providing a potential mechanism for tumor sensitization to TMZ by CBD. Conclusion These data support the potential therapeutic benefits of using CBD to enhance the antitumor activity of TMZ in GBM patients.