A platform for rapid patient-derived cutaneous neurofibroma organoid establishment and screening.

Localized cutaneous neurofibromas (cNFs) are benign tumors that arise in the dermis of patients affected by neurofibromatosis type 1 syndrome. cNFs are benign lesions: they do not undergo malignant transformation or metastasize. Nevertheless, they can cover a significant proportion of the body, with some individuals developing hundreds to thousands of lesions. cNFs can cause pain, itching, and disfigurement resulting in substantial socio-emotional repercussions. Currently, surgery and laser desiccation are the sole treatment options but may result in scarring and potential regrowth from incomplete removal. To identify effective systemic therapies, we introduce an approach to establish and screen cNF organoids. We optimized conditions to support the ex vivo growth of genomically diverse cNFs. Patient-derived cNF organoids closely recapitulate cellular and molecular features of parental tumors as measured by immunohistopathology, methylation, RNA sequencing, and flow cytometry. Our cNF organoid platform enables rapid screening of hundreds of compounds in a patient- and tumor-specific manner.

The TLK-ASF1 histone chaperone pathway plays a critical role in IL-1b-mediated AML progression.

Identifying and targeting microenvironment-driven pathways that are active across acute myeloid leukemia (AML) genetic subtypes should allow the development of more broadly effective therapies. The pro-inflammatory cytokine IL-1 is abundant in the AML microenvironment and promotes leukemic growth. Through RNA-sequencing analysis, we identify that IL-1 upregulated ASF1B (anti-silencing function-1B), a histone chaperone, in AML progenitors compared to healthy progenitors. ASF1B, along with its paralogous protein ASF1A recruits H3-H4 histones onto the replication fork during S-phase, a process regulated by tousled-like kinase 1 and 2 (TLKs). While ASF1s and TLKs are known to be overexpressed in multiple solid tumors and associated with poor prognosis, their functional roles in hematopoiesis and inflammation-driven leukemia remain unexplored. In this study, we identify that ASF1s and TLKs are over-expressed in multiple genetic subtypes of AML. We demonstrate that depletion of ASF1s significantly reduces leukemic cell growth in both in vitro and in vivo models using human cells. Using a murine model we show that overexpression of ASF1B accelerates leukemia progression. Moreover, Asf1b or Tlk2 deletion delayed leukemia progression while these proteins are dispensable for normal hematopoiesis. Through proteomics and phosphoproteomics analyses, we uncover that the TLK-ASF1 pathway promotes leukemogenesis by impacting the cell cycle and DNA damage pathways. Collectively, our findings identify the TLK1-ASF1 pathway as a novel mediator of inflammatory signaling and a promising therapeutic target for AML treatment across diverse genetic subtypes. Selective inhibition of this pathway offers potential opportunities to intervene effectively, address intratumoral heterogeneity, and ultimately improve clinical outcomes in AML.

Targeting CCL2/CCR2 Signaling Overcomes MEK Inhibitor Resistance in Acute Myeloid Leukemia.

PURPOSE: Emerging evidence underscores the critical role of extrinsic factors within the microenvironment in protecting leukemia cells from therapeutic interventions, driving disease progression, and promoting drug resistance in acute myeloid leukemia (AML). This finding emphasizes the need for the identification of targeted therapies that inhibit intrinsic and extrinsic signaling to overcome drug resistance in AML. EXPERIMENTAL DESIGN: We performed a comprehensive analysis utilizing a cohort of ∼300 AML patient samples. This analysis encompassed the evaluation of secreted cytokines/growth factors, gene expression, and ex vivo drug sensitivity to small molecules. Our investigation pinpointed a notable association between elevated levels of CCL2 and diminished sensitivity to the MEK inhibitors (MEKi). We validated this association through loss-of-function and pharmacologic inhibition studies. Further, we deployed global phosphoproteomics and CRISPR/Cas9 screening to identify the mechanism of CCR2-mediated MEKi resistance in AML. RESULTS: Our multifaceted analysis unveiled that CCL2 activates multiple prosurvival pathways, including MAPK and cell-cycle regulation in MEKi-resistant cells. Employing combination strategies to simultaneously target these pathways heightened growth inhibition in AML cells. Both genetic and pharmacologic inhibition of CCR2 sensitized AML cells to trametinib, suppressing proliferation while enhancing apoptosis. These findings underscore a new role for CCL2 in MEKi resistance, offering combination therapies as an avenue to circumvent this resistance. CONCLUSIONS: Our study demonstrates a compelling rationale for translating CCL2/CCR2 axis inhibitors in combination with MEK pathway-targeting therapies, as a potent strategy for combating drug resistance in AML. This approach has the potential to enhance the efficacy of treatments to improve AML patient outcomes.

Decomprolute is a benchmarking platform designed for multiomics-based tumor deconvolution.

Tumor deconvolution enables the identification of diverse cell types that comprise solid tumors. To date, however, both the algorithms developed to deconvolve tumor samples, and the gold-standard datasets used to assess the algorithms are geared toward the analysis of gene expression (e.g., RNA sequencing) rather than protein levels. Despite the popularity of gene expression datasets, protein levels often provide a more accurate view of rare cell types. To facilitate the use, development, and reproducibility of multiomic deconvolution algorithms, we introduce Decomprolute, a Common Workflow Language framework that leverages containerization to compare tumor deconvolution algorithms across multiomic datasets. Decomprolute incorporates the large-scale multiomic datasets produced by the Clinical Proteomic Tumor Analysis Consortium (CPTAC), which include matched mRNA expression and proteomic data from thousands of tumors across multiple cancer types to build a fully open-source, containerized proteogenomic tumor deconvolution benchmarking platform. http://pnnl-compbio.github.io/decomprolute.

Pan-cancer proteogenomics characterization of tumor immunity.

Despite the successes of immunotherapy in cancer treatment over recent decades, less than <10%-20% cancer cases have demonstrated durable responses from immune checkpoint blockade. To enhance the efficacy of immunotherapies, combination therapies suppressing multiple immune evasion mechanisms are increasingly contemplated. To better understand immune cell surveillance and diverse immune evasion responses in tumor tissues, we comprehensively characterized the immune landscape of more than 1,000 tumors across ten different cancers using CPTAC pan-cancer proteogenomic data. We identified seven distinct immune subtypes based on integrative learning of cell type compositions and pathway activities. We then thoroughly categorized unique genomic, epigenetic, transcriptomic, and proteomic changes associated with each subtype. Further leveraging the deep phosphoproteomic data, we studied kinase activities in different immune subtypes, which revealed potential subtype-specific therapeutic targets. Insights from this work will facilitate the development of future immunotherapy strategies and enhance precision targeting with existing agents.

Mapping the proteogenomic landscape enables prediction of drug response in acute myeloid leukemia.

Acute myeloid leukemia is a poor-prognosis cancer commonly stratified by genetic aberrations, but these mutations are often heterogeneous and fail to consistently predict therapeutic response. Here, we combine transcriptomic, proteomic, and phosphoproteomic datasets with ex vivo drug sensitivity data to help understand the underlying pathophysiology of AML beyond mutations. We measure the proteome and phosphoproteome of 210 patients and combine them with genomic and transcriptomic measurements to identify four proteogenomic subtypes that complement existing genetic subtypes. We build a predictor to classify samples into subtypes and map them to a "landscape" that identifies specific drug response patterns. We then build a drug response prediction model to identify drugs that target distinct subtypes and validate our findings on cell lines representing various stages of quizartinib resistance. Our results show how multiomics data together with drug sensitivity data can inform therapy stratification and drug combinations in AML.

Mass Spectrometry-Based Proteogenomics: New Therapeutic Opportunities for Precision Medicine.

Proteogenomics refers to the integration of comprehensive genomic, transcriptomic, and proteomic measurements from the same samples with the goal of fully understanding the regulatory processes converting genotypes to phenotypes, often with an emphasis on gaining a deeper understanding of disease processes. Although specific genetic mutations have long been known to drive the development of multiple cancers, gene mutations alone do not always predict prognosis or response to targeted therapy. The benefit of proteogenomics research is that information obtained from proteins and their corresponding pathways provides insight into therapeutic targets that can complement genomic information by providing an additional dimension regarding the underlying mechanisms and pathophysiology of tumors. This review describes the novel insights into tumor biology and drug resistance derived from proteogenomic analysis while highlighting the clinical potential of proteogenomic observations and advances in technique and analysis tools.

CDK4/6 inhibition enhances SHP2 inhibitor efficacy and is dependent upon RB function in malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive soft tissue sarcomas with limited treatment options, and new effective therapeutic strategies are desperately needed. We observe antiproliferative potency of genetic depletion of PTPN11 or pharmacological inhibition using the SHP2 inhibitor (SHP2i) TNO155. Our studies into the signaling response to SHP2i reveal that resistance to TNO155 is partially mediated by reduced RB function, and we therefore test the addition of a CDK4/6 inhibitor (CDK4/6i) to enhance RB activity and improve TNO155 efficacy. In combination, TNO155 attenuates the adaptive response to CDK4/6i, potentiates its antiproliferative effects, and converges on enhancement of RB activity, with greater suppression of cell cycle and inhibitor-of-apoptosis proteins, leading to deeper and more durable antitumor activity in in vitro and in vivo patient-derived models of MPNST, relative to either single agent. Overall, our study provides timely evidence to support the clinical advancement of this combination strategy in patients with MPNST and other tumors driven by loss of NF1.

Proteome Mapping of the Human Pancreatic Islet Microenvironment Reveals Endocrine-Exocrine Signaling Sphere of Influence.

The need for a clinically accessible method with the ability to match protein activity within heterogeneous tissues is currently unmet by existing technologies. Our proteomics sample preparation platform, named microPOTS (Microdroplet Processing in One pot for Trace Samples), can be used to measure relative protein abundance in micron-scale samples alongside the spatial location of each measurement, thereby tying biologically interesting proteins and pathways to distinct regions. However, given the smaller pixel/voxel number and amount of tissue measured, standard mass spectrometric analysis pipelines have proven inadequate. Here we describe how existing computational approaches can be adapted to focus on the specific biological questions asked in spatial proteomics experiments. We apply this approach to present an unbiased characterization of the human islet microenvironment comprising the entire complex array of cell types involved while maintaining spatial information and the degree of the islet's sphere of influence. We identify specific functional activity unique to the pancreatic islet cells and demonstrate how far their signature can be detected in the adjacent tissue. Our results show that we can distinguish pancreatic islet cells from the neighboring exocrine tissue environment, recapitulate known biological functions of islet cells, and identify a spatial gradient in the expression of RNA processing proteins within the islet microenvironment.

Ex vivo to in vivo model of malignant peripheral nerve sheath tumors for precision oncology.

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft tissue sarcomas that often develop in patients with neurofibromatosis type 1 (NF1). To address the critical need for novel therapeutics in MPNST, we aimed to establish an ex vivo 3D platform that accurately captured the genomic diversity of MPNST and could be utilized in a medium-throughput manner for drug screening studies to be validated in vivo using patient-derived xenografts (PDX). METHODS: Genomic analysis was performed on all PDX-tumor pairs. Selected PDX were harvested for assembly into 3D microtissues. Based on prior work in our labs, we evaluated drugs (trabectedin, olaparib, and mirdametinib) ex vivo and in vivo. For 3D microtissue studies, cell viability was the endpoint as assessed by Zeiss Axio Observer. For PDX drug studies, tumor volume was measured twice weekly. Bulk RNA sequencing was performed to identify pathways enriched in cells. RESULTS: We developed 13 NF1-associated MPNST-PDX and identified mutations or structural abnormalities in NF1 (100%), SUZ12 (85%), EED (15%), TP53 (15%), CDKN2A (85%), and chromosome 8 gain (77%). We successfully assembled PDX into 3D microtissues, categorized as robust (>90% viability at 48 h), good (>50%), or unusable (<50%). We evaluated drug response to "robust" or "good" microtissues, namely MN-2, JH-2-002, JH-2-079-c, and WU-225. Drug response ex vivo predicted drug response in vivo, and enhanced drug effects were observed in select models. CONCLUSIONS: These data support the successful establishment of a novel 3D platform for drug discovery and MPNST biology exploration in a system representative of the human condition.

The Superfund Research Program Analytics Portal: linking environmental chemical exposure to biological phenotypes.

The OSU/PNNL Superfund Research Program (SRP) represents a longstanding collaboration to quantify Polycyclic Aromatic Hydrocarbons (PAHs) at various superfund sites in the Pacific Northwest and assess their potential impact on human health. To link the chemical measurements to biological activity, we describe the use of the zebrafish as a high-throughput developmental toxicity model that provides quantitative measurements of the exposure to chemicals. Toward this end, we have linked over 150 PAHs found at Superfund sites to the effect of these same chemicals in zebrafish, creating a rich dataset that links environmental exposure to biological response. To quantify this response, we have implemented a dose-response modelling pipeline to calculate benchmark dose parameters which enable potency comparison across over 500 chemicals and 12 of the phenotypes measured in zebrafish. We provide a rich dataset for download and analysis as well as a web portal that provides public access to this dataset via an interactive web site designed to support exploration and re-use of these data by the scientific community at http://srp.pnnl.gov .

CDK4/6 inhibition enhances SHP2 inhibitor efficacy and is dependent upon restoration of RB function in malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive soft tissue sarcomas with limited treatment options, and novel effective therapeutic strategies are desperately needed. We observe anti-proliferative efficacy of genetic depletion or pharmacological inhibition using the clinically available SHP2 inhibitor (SHP2i) TNO155. Our studies into the signaling response to SHP2i reveal that resistance to TNO155 is partially mediated by reduced RB function, and we therefore test the addition of a CDK4/6 inhibitor (CDK4/6i) to enhance RB activity and improve TNO155 efficacy. In combination, TNO155 attenuates the adaptive response to CDK4/6i, potentiates its anti-proliferative effects, and converges on enhancement of RB activity, with greater suppression of cell cycle and inhibitor-of-apoptosis proteins, leading to deeper and more durable anti-tumor activity in in vitro and in vivo patient-derived models of MPNST, relative to either single agent. Overall, our study provides timely evidence to support the clinical advancement of this combination strategy in patients with MPNST and other tumors driven by loss of NF1.

Proteomic and phosphoproteomic measurements enhance ability to predict ex vivo drug response in AML.

Acute Myeloid Leukemia (AML) affects 20,000 patients in the US annually with a five-year survival rate of approximately 25%. One reason for the low survival rate is the high prevalence of clonal evolution that gives rise to heterogeneous sub-populations of leukemic cells with diverse mutation spectra, which eventually leads to disease relapse. This genetic heterogeneity drives the activation of complex signaling pathways that is reflected at the protein level. This diversity makes it difficult to treat AML with targeted therapy, requiring custom patient treatment protocols tailored to each individual's leukemia. Toward this end, the Beat AML research program prospectively collected genomic and transcriptomic data from over 1000 AML patients and carried out ex vivo drug sensitivity assays to identify genomic signatures that could predict patient-specific drug responses. However, there are inherent weaknesses in using only genetic and transcriptomic measurements as surrogates of drug response, particularly the absence of direct information about phosphorylation-mediated signal transduction. As a member of the Clinical Proteomic Tumor Analysis Consortium, we have extended the molecular characterization of this cohort by collecting proteomic and phosphoproteomic measurements from a subset of these patient samples (38 in total) to evaluate the hypothesis that proteomic signatures can improve the ability to predict response to 26 drugs in AML ex vivo samples. In this work we describe our systematic, multi-omic approach to evaluate proteomic signatures of drug response and compare protein levels to other markers of drug response such as mutational patterns. We explore the nuances of this approach using two drugs that target key pathways activated in AML: quizartinib (FLT3) and trametinib (Ras/MEK), and show how patient-derived signatures can be interpreted biologically and validated in cell lines. In conclusion, this pilot study demonstrates strong promise for proteomics-based patient stratification to assess drug sensitivity in AML.

A field guide to cultivating computational biology.

Evolving in sync with the computation revolution over the past 30 years, computational biology has emerged as a mature scientific field. While the field has made major contributions toward improving scientific knowledge and human health, individual computational biology practitioners at various institutions often languish in career development. As optimistic biologists passionate about the future of our field, we propose solutions for both eager and reluctant individual scientists, institutions, publishers, funding agencies, and educators to fully embrace computational biology. We believe that in order to pave the way for the next generation of discoveries, we need to improve recognition for computational biologists and better align pathways of career success with pathways of scientific progress. With 10 outlined steps, we call on all adjacent fields to move away from the traditional individual, single-discipline investigator research model and embrace multidisciplinary, data-driven, team science.

The AML microenvironment catalyzes a stepwise evolution to gilteritinib resistance.

Our study details the stepwise evolution of gilteritinib resistance in FLT3-mutated acute myeloid leukemia (AML). Early resistance is mediated by the bone marrow microenvironment, which protects residual leukemia cells. Over time, leukemia cells evolve intrinsic mechanisms of resistance, or late resistance. We mechanistically define both early and late resistance by integrating whole-exome sequencing, CRISPR-Cas9, metabolomics, proteomics, and pharmacologic approaches. Early resistant cells undergo metabolic reprogramming, grow more slowly, and are dependent upon Aurora kinase B (AURKB). Late resistant cells are characterized by expansion of pre-existing NRAS mutant subclones and continued metabolic reprogramming. Our model closely mirrors the timing and mutations of AML patients treated with gilteritinib. Pharmacological inhibition of AURKB resensitizes both early resistant cell cultures and primary leukemia cells from gilteritinib-treated AML patients. These findings support a combinatorial strategy to target early resistant AML cells with AURKB inhibitors and gilteritinib before the expansion of pre-existing resistance mutations occurs.

leapR: An R Package for Multiomic Pathway Analysis.

A generalized goal of many high-throughput data studies is to identify functional mechanisms that underlie observed biological phenomena, whether they be disease outcomes or metabolic output. Increasingly, studies that rely on multiple sources of high-throughput data (genomic, transcriptomic, proteomic, metabolomic) are faced with a challenge of summarizing the data to generate testable hypotheses. However, this requires a time-consuming process to evaluate numerous statistical methods across numerous data sources. Here, we introduce the leapR package, a framework to rapidly assess biological pathway activity using diverse statistical tests and data sources, allowing facile integration of multisource data. The leapR package with a user manual and example workflow is available for download from GitHub (https://github.com/biodataganache/leapR).

Knowledge-based classification of fine-grained immune cell types in single-cell RNA-Seq data.

Single-cell RNA sequencing (scRNA-Seq) is an emerging strategy for characterizing immune cell populations. Compared to flow or mass cytometry, scRNA-Seq could potentially identify cell types and activation states that lack precise cell surface markers. However, scRNA-Seq is currently limited due to the need to manually classify each immune cell from its transcriptional profile. While recently developed algorithms accurately annotate coarse cell types (e.g. T cells versus macrophages), making fine distinctions (e.g. CD8+ effector memory T cells) remains a difficult challenge. To address this, we developed a machine learning classifier called ImmClassifier that leverages a hierarchical ontology of cell type. We demonstrate that its predictions are highly concordant with flow-based markers from CITE-seq and outperforms other tools (+15% recall, +14% precision) in distinguishing fine-grained cell types with comparable performance on coarse ones. Thus, ImmClassifier can be used to explore more deeply the heterogeneity of the immune system in scRNA-Seq experiments.

Integrated molecular and clinical analysis of low-grade gliomas in children with neurofibromatosis type 1 (NF1).

Low-grade gliomas (LGGs) are the most common childhood brain tumor in the general population and in individuals with the Neurofibromatosis type 1 (NF1) cancer predisposition syndrome. Surgical biopsy is rarely performed prior to treatment in the setting of NF1, resulting in a paucity of tumor genomic information. To define the molecular landscape of NF1-associated LGGs (NF1-LGG), we integrated clinical data, histological diagnoses, and multi-level genetic/genomic analyses on 70 individuals from 25 centers worldwide. Whereas, most tumors harbored bi-allelic NF1 inactivation as the only genetic abnormality, 11% had additional mutations. Moreover, tumors classified as non-pilocytic astrocytoma based on DNA methylation analysis were significantly more likely to harbor these additional mutations. The most common secondary alteration was FGFR1 mutation, which conferred an additional growth advantage in multiple complementary experimental murine Nf1 models. Taken together, this comprehensive characterization has important implications for the management of children with NF1-LGG, distinct from their sporadic counterparts.

Chromosome 8 gain is associated with high-grade transformation in MPNST.

One of the most common malignancies affecting adults with Neurofibromatosis type 1 (NF1) is the malignant peripheral nerve sheath tumor (MPNST), an aggressive and often fatal sarcoma that commonly arises from benign plexiform neurofibromas. Despite advances in our understanding of MPNST pathobiology, there are few effective therapeutic options, and no investigational agents have proven successful in clinical trials. To further understand the genomic heterogeneity of MPNST, and to generate a preclinical platform that encompasses this heterogeneity, we developed a collection of NF1-MPNST patient-derived xenografts (PDX). These PDX were compared with the primary tumors from which they were derived using copy number analysis, whole exome sequencing, and RNA sequencing. We identified chromosome 8 gain as a recurrent genomic event in MPNST and validated its occurrence by FISH in the PDX and parental tumors, in a validation cohort, and by single-cell sequencing in the PDX. Finally, we show that chromosome 8 gain is associated with inferior overall survival in soft-tissue sarcomas. These data suggest that chromosome 8 gain is a critical event in MPNST pathogenesis and may account for the aggressive nature and poor outcomes in this sarcoma subtype.

A clinically and genomically annotated nerve sheath tumor biospecimen repository.

Nerve sheath tumors occur as a heterogeneous group of neoplasms in patients with neurofibromatosis type 1 (NF1). The malignant form represents the most common cause of death in people with NF1, and even when benign, these tumors can result in significant disfigurement, neurologic dysfunction, and a range of profound symptoms. Lack of human tissue across the peripheral nerve tumors common in NF1 has been a major limitation in the development of new therapies. To address this unmet need, we have created an annotated collection of patient tumor samples, patient-derived cell lines, and patient-derived xenografts, and carried out high-throughput genomic and transcriptomic characterization to serve as a resource for further biologic and preclinical therapeutic studies. In this work, we release genomic and transcriptomic datasets comprised of 55 tumor samples derived from 23 individuals, complete with clinical annotation. All data are publicly available through the NF Data Portal and at http://synapse.org/jhubiobank.