Siva Kumar Natarajan | University of Michigan (original) (raw)

Papers by Siva Kumar Natarajan

Cancer Research

Ependymomas are fatal brain malignancies with very few treatment options. More than 70% of suprat... more Ependymomas are fatal brain malignancies with very few treatment options. More than 70% of supratentorial (ST) ependymomas harbor fusions of the zinc-finger containing, chromatin modifier ZFTA and the transcriptional activator of NF-κB signaling, RELA. Oncogene-driven metabolic reprogramming is a fundamental hallmark of cancer that enables sustained tumor proliferation. ZFTA-RELA fusion protein is essential for tumorigenesis and our goal is to determine how it drives metabolism in ST-ependymomas. To address this, we developed an in vitro isogenic system by expressing the ZFTA-RELA fusion protein in immortalized mouse neural stem cells (ZFTA-RELAFUS). Using this system, we show that these tumor cells selectively upregulate expression of the glutamine transporter (SLC1A5), glutaminase (GLS), and many downstream enzymes in the glutamine metabolic pathway. Therefore, we hypothesized that the ZFTA-RELA fusion drives glutamine metabolism in ST-ependymomas. We further demonstrate that ZFTA...

Science Translational Medicine

Description Childhood posterior fossa group A ependymomas demonstrate integrated metabolic and ep... more Description Childhood posterior fossa group A ependymomas demonstrate integrated metabolic and epigenetic pathways that can be disrupted by metformin. Understanding childhood ependymomas Children with posterior fossa group A (PFA) ependymomas have a dismal prognosis. PFAs overexpress the protein EZHIP (EZH inhibitory protein), and Panwalkar and colleagues investigated whether EZHIP could epigenetically rewire metabolic pathways. Glycolysis and tricarboxylic acid (TCA) cycle metabolism were enhanced in tumors with high EZHIP expression, associated with enrichment of histone H3 lysine 27 acetylation at hexokinase-2, pyruvate dehydrogenase, and AMPKα-2. The antidiabetic drug metformin, an AMPK activator, altered histone marks, lowered EZHIP concentrations, and suppressed TCA cycle metabolism, with decreased tumor volumes and increased survival in mice carrying patient-derived xenografts. These findings suggest that targeting integrated epigenetic and metabolomic pathways could be a therapeutic strategy for children with PFAs. Childhood posterior fossa group A ependymomas (PFAs) have limited treatment options and bear dismal prognoses compared to group B ependymomas (PFBs). PFAs overexpress the oncohistone-like protein EZHIP (enhancer of Zeste homologs inhibitory protein), causing global reduction of repressive histone H3 lysine 27 trimethylation (H3K27me3), similar to the oncohistone H3K27M. Integrated metabolic analyses in patient-derived cells and tumors, single-cell RNA sequencing of tumors, and noninvasive metabolic imaging in patients demonstrated enhanced glycolysis and tricarboxylic acid (TCA) cycle metabolism in PFAs. Furthermore, high glycolytic gene expression in PFAs was associated with a poor outcome. PFAs demonstrated high EZHIP expression associated with poor prognosis and elevated activating mark histone H3 lysine 27 acetylation (H3K27ac). Genomic H3K27ac was enriched in PFAs at key glycolytic and TCA cycle–related genes including hexokinase-2 and pyruvate dehydrogenase. Similarly, mouse neuronal stem cells (NSCs) expressing wild-type EZHIP (EZHIP-WT) versus catalytically attenuated EZHIP-M406K demonstrated H3K27ac enrichment at hexokinase-2 and pyruvate dehydrogenase, accompanied by enhanced glycolysis and TCA cycle metabolism. AMPKα-2, a key component of the metabolic regulator AMP-activated protein kinase (AMPK), also showed H3K27ac enrichment in PFAs and EZHIP-WT NSCs. The AMPK activator metformin lowered EZHIP protein concentrations, increased H3K27me3, suppressed TCA cycle metabolism, and showed therapeutic efficacy in vitro and in vivo in patient-derived PFA xenografts in mice. Our data indicate that PFAs and EZHIP-WT–expressing NSCs are characterized by enhanced glycolysis and TCA cycle metabolism. Repurposing the antidiabetic drug metformin lowered pathogenic EZHIP, increased H3K27me3, and suppressed tumor growth, suggesting that targeting integrated metabolic/epigenetic pathways is a potential therapeutic strategy for treating childhood ependymomas.

Science Translational Medicine

Description Histone H3.3G34R/V mutant gliomas demonstrate LIF/STAT pathway activation that can be... more Description Histone H3.3G34R/V mutant gliomas demonstrate LIF/STAT pathway activation that can be disrupted for therapeutic benefit. STAT3 inhibition for H3.3G34R/V gliomas High-grade gliomas with arginine or valine substitutions at the glycine-34 residue on histone H3.3 (H3.3G34R/V gliomas) carry a poor prognosis. Here, Sweha and colleagues investigated the epigenetics of these tumors, finding activating epigenetic modifications on histone H3 lysine residues, as well as DNA promoter hypomethylation. They also found redistribution of repressive histone marks at the leukemia inhibitory factor (LIF) locus, leading to increased LIF abundance and secretion. LIF activated STAT3 signaling, which the authors targeted with a blood-brain barrier–permeable small-molecule inhibitor called WP1066. When given to mice carrying H3.3G34R/V tumors, WP1066 suppressed tumor growth, suggesting that inhibition of STAT3 signaling may be of therapeutic value in H3.3G34R/V gliomas. High-grade gliomas with arginine or valine substitutions of the histone H3.3 glycine-34 residue (H3.3G34R/V) carry a dismal prognosis, and current treatments, including radiotherapy and chemotherapy, are not curative. Because H3.3G34R/V mutations reprogram epigenetic modifications, we undertook a comprehensive epigenetic approach using ChIP sequencing and ChromHMM computational analysis to define therapeutic dependencies in H3.3G34R/V gliomas. Our analyses revealed a convergence of epigenetic alterations, including (i) activating epigenetic modifications on histone H3 lysine (K) residues such as H3K36 trimethylation (H3K36me3), H3K27 acetylation (H3K27ac), and H3K4 trimethylation (H3K4me3); (ii) DNA promoter hypomethylation; and (iii) redistribution of repressive histone H3K27 trimethylation (H3K27me3) to intergenic regions at the leukemia inhibitory factor (LIF) locus to drive increased LIF abundance and secretion by H3.3G34R/V cells. LIF activated signal transducer and activator of transcription 3 (STAT3) signaling in an autocrine/paracrine manner to promote survival of H3.3G34R/V glioma cells. Moreover, immunohistochemistry and single-cell RNA sequencing from H3.3G34R/V patient tumors revealed high STAT3 protein and RNA expression, respectively, in tumor cells with both inter- and intratumor heterogeneity. We targeted STAT3 using a blood-brain barrier–penetrable small-molecule inhibitor, WP1066, currently in clinical trials for adult gliomas. WP1066 treatment resulted in H3.3G34R/V tumor cell toxicity in vitro and tumor suppression in preclinical mouse models established with KNS42 cells, SJ-HGGx42-c cells, or in utero electroporation techniques. Our studies identify the LIF/STAT3 pathway as a key epigenetically driven and druggable vulnerability in H3.3G34R/V gliomas. This finding could inform development of targeted, combination therapies for these lethal brain tumors.

Cancer Research

This study uncovers a molecular mechanism linking drug-induced resistance and tumor immunity and ... more This study uncovers a molecular mechanism linking drug-induced resistance and tumor immunity and provides novel engineered solutions that target these mechanisms in the tumor and improve immunity, thus mitigating off-target effects. Drug-induced resistance, or tolerance, is an emerging yet poorly understood failure of anticancer therapy. The interplay between drug-tolerant cancer cells and innate immunity within the tumor, the consequence on tumor growth, and therapeutic strategies to address these challenges remain undescribed. Here, we elucidate the role of taxane-induced resistance on natural killer (NK) cell tumor immunity in triple-negative breast cancer (TNBC) and the design of spatiotemporally controlled nanomedicines, which boost therapeutic efficacy and invigorate “disabled” NK cells. Drug tolerance limited NK cell immune surveillance via drug-induced depletion of the NK-activating ligand receptor axis, NK group 2 member D, and MHC class I polypeptide-related sequence A, B. Systems biology supported by empirical evidence revealed the heat shock protein 90 (Hsp90) simultaneously controls immune surveillance and persistence of drug-treated tumor cells. On the basis of this evidence, we engineered a “chimeric” nanotherapeutic tool comprising taxanes and a cholesterol-tethered Hsp90 inhibitor, radicicol, which targets the tumor, reduces tolerance, and optimally reprimes NK cells via prolonged induction of NK-activating ligand receptors via temporal control of drug release in vitro and in vivo. A human ex vivo TNBC model confirmed the importance of NK cells in drug-induced death under pressure of clinically approved agents. These findings highlight a convergence between drug-induced resistance, the tumor immune contexture, and engineered approaches that consider the tumor and microenvironment to improve the success of combinatorial therapy. Significance: This study uncovers a molecular mechanism linking drug-induced resistance and tumor immunity and provides novel engineered solutions that target these mechanisms in the tumor and improve immunity, thus mitigating off-target effects.

Cellular and Molecular Bioengineering

Introduction-Immune checkpoint inhibitors that boost cytotoxic T cell-based immune responses have... more Introduction-Immune checkpoint inhibitors that boost cytotoxic T cell-based immune responses have emerged as one of the most promising approaches in cancer treatment. However, it is increasingly being realized that T cell activation needs to be rationally combined with molecularly targeted therapeutics for a maximal anti-tumor outcome. Currently, two oncogenic drivers, MAPK and PI3K-mTOR have emerged as the two main molecular targets for combining with immunotherapy. However, there are major challenges in enabling such combinations: first, such combinations can result in high rates of toxicity. Second, while, these molecular targets could be driving tumor progression, they are essential for activation of the immune cells. So, the kinase inhibitors and immunotherapy can antagonize each other. Objectives-We rationalized that the synergistic combination of kinase inhibitors and immunotherapy could be enabled by dual inhibitors-loaded supramolecular nanotherapeutics (DiLN) that can co-deliver PI3K-and MAPK-inhibitors to the cancer cells and activate immune response by T cellmodulating immunotherapy, resulting in greater anti-tumor efficacy while minimizing toxicity. Methods-We engineered DiLNs by designing the amphiphilic building blocks (both drugs and co-lipids) that enables supramolecular nanoassembly. DiLNs were tested for their physiochemical properties including size, morphology, stability and drug release kinetics profiles. The efficacy of DiLNs was tested in drug-resistant cells such as BRAF V600E melanoma (D4M), Clear cell ovarian carcinoma (TOV21G) cells. The tumor inhibition efficiency of DiLNs in combination with immune checkpoint inhibitor antibody was studied in syngeneic D4M animal model. Results-DiLNs were stable for over a month and released the drugs in a sustained manner. In vitro cytotoxicity studies in D4M and TOV21G cells showed that DiLNs were significantly more effective than free drugs. In vivo studies showed that the combination of DiLNs with anti PD-L1 antibody resulted in superior antitumor effect and survival. Conclusion-This study shows that the rational combination of DiLNs that target multiple oncogenic signaling pathways with immune checkpoint inhibitors could emerge as an effective strategy to improve immunotherapeutic response against drug resistant tumors.

Nature Biomedical Engineering

Effectively activating macrophages that can 'eat' cancer cells is challenging. In particular, can... more Effectively activating macrophages that can 'eat' cancer cells is challenging. In particular, cancer cells secrete macrophage colony stimulating factor (MCSF), which polarizes tumour-associated macrophages from an antitumour M1 phenotype to a pro-tumorigenic M2 phenotype. Also, cancer cells can express CD47, a 'don't eat me' signal that ligates with the signal regulatory protein alpha (SIRPα) receptor on macrophages to prevent phagocytosis. Here, we show that a supramolecular assembly consisting of amphiphiles inhibiting the colony stimulating factor 1 receptor (CSF-1R) and displaying SIRPα-blocking antibodies with a drug-to-antibody ratio of 17,000 can disable both mechanisms. The supramolecule homes onto SIRPα on macrophages, blocking the CD47-SIRPα signalling axis while sustainedly inhibiting CSF-1R. The supramolecule enhances M2-to-M1 repolarization within the tumour microenvironment, and significantly improves antitumour and antimetastatic efficacies in two aggressive animal models of melanoma and breast cancer, with respect to clinically available small-molecule and biologic inhibitors of CSF-1R signalling. Simultaneously blocking the CD47-SIRPα and MCSF-CSF-1R signalling axes may constitute a promising immunotherapy.

Acta neuropathologica, 2018

Diffuse midline gliomas harboring a recurrent H3 lysine 27-to-methionine (p.Lys27Met, H3K27M) con... more Diffuse midline gliomas harboring a recurrent H3 lysine 27-to-methionine (p.Lys27Met, H3K27M) constitute a recently defined pathologic entity with a particularly poor prognosis. They mainly occur in midline structures, such as the pons and thalamus of children and young adults, and are highly infiltrative [2]. More recently, case reports have described the H3K27M mutation in circumscribed (non-diffuse) gliomas, many of which are low-grade (e.g., pilocytic astrocytoma, ganglioglioma) [1, 3–5] (additional references are provided in Online Resource 1). Because of the rarity of these tumors, it is unknown whether they carry the poor clinical outcome ascribed to H3K27M-mutant infiltrating gliomas of the midline. Here, we address this gap in our knowledge by performing an integrated meta-analysis on collated clinical and pathologic data from published studies, data repositories, and collaborative efforts. A systematic search of the literature was performed from 2012 to November, 2017. Tumors were categorized based on diagnosis, location, histologic grade, growth pattern, and H3K27M mutation status. IDH 1/2-mutant diffuse gliomas were excluded to avoid bias arising from the good prognosis attributed to this mutation. Circumscribed gliomas included pilocytic astrocytoma, ganglioglioma/glioneuronal tumor/ ganglion cell tumor, pleomorphic xanthoastrocytoma, and ependymoma. Data from The Cancer Genome Atlas (TCGA) (http ://canc erge nome .nih.gov/) were obtained through the cBioPortal and PedcBioPortal for Cancer Genomics. Factors extracted included age, sex, overall survival, tumor location, histopathologic diagnosis, and WHO grade. Patient samples were then cross-referenced across studies to filter out duplicate data. Cases were also acquired from our inhouse sequencing efforts and through collaboration with the Mayo Clinic in Rochester, Minnesota. Co-occurring mutations were also documented. The endpoint extracted from all data sources was overall survival (OS). Survival functions were estimated using the Kaplan–Meier method and differences analyzed with the log-rank (Mantel–Cox) test using GraphPad Prism software (version 7). The Coxproportional hazards model was used to calculate hazard ratios (HR) using SPSS (version 24, IBM). All studies were approved by ethics committees at the respective institutions (see Online Resource 1 for further details of the methods). Among published studies and through institutional collaboration, we identified 28 cases of H3K27M-mutant circumscribed gliomas (grade I: n = 19; grade III, n = 9). Histopathology included pilocytic astrocytoma (n = 7), ganglioglioma (n = 10), anaplastic ganglioglioma (n = 3), glioneuronal tumor (n = 1), anaplastic glioneuronal tumor (n = 1), ganglion cell tumor (n = 1; Fig. 1a–d), anaplastic ependymoma (n = 3), and circumscribed glioma, not further specified (n = 2) (see Online Resource 2 for references and case details). Strikingly, more than 96% (n = 26/27 cases with location provided) of H3K27M-mutant circumscribed gliomas occurred within the midline, including the brainstem (n = 7), thalamus (n = 5), cerebellum (n = 2), spinal cord (n = 8), and other midline regions (peduncle, posterior fossa, and midline-not further specified; n = 4) (Fig. 1g). Of the remaining two cases, one was reported in the cerebrum, while the location was not specified for the other. H3K27M mutations occurred in H3F3A (n = 21), HIST1H3B (n = 1), Electronic supplementary material The online version of this article (http s://doi.org/10.1007 /s004 01-018-1805 -3) contains supplementary material, which is available to authorized users.

Cancer Research

Epigenetic modifications including altered DNA methylation and histone posttranslational modifica... more Epigenetic modifications including altered DNA methylation and histone posttranslational modifications (PTM) are central to the biology of several cancers. These modifications can regulate DNA accessibility and consequently, gene expression. In this issue, Wojcik and colleagues explore epigenetic drivers of malignant peripheral nerve sheath tumors (MPNST) harboring loss-of-function polycomb-repressive complex 2 mutations. They demonstrate alterations in specific histone PTMs and a global increase in DNA methylation. Notably, epigenetic alterations related with aberrant upregulation of proteins involved in immune evasion, which informed identification of potential therapeutic vulnerabilities. This study helps understand the complex biology of MPNSTs and may enable future therapeutic development.See related article by Wojcik et al., p. 3205.

Cancers

Altered metabolism is a hallmark of cancer cells. Tumor cells rewire their metabolism to support ... more Altered metabolism is a hallmark of cancer cells. Tumor cells rewire their metabolism to support their uncontrolled proliferation by taking up nutrients from the microenvironment. The amino acid glutamine is a key nutrient that fuels biosynthetic processes including ATP generation, redox homeostasis, nucleotide, protein, and lipid synthesis. Glutamine as a precursor for the neurotransmitter glutamate, and plays a critical role in the normal functioning of the brain. Brain tumors that grow in this glutamine/glutamate rich microenvironment can make synaptic connections with glutamatergic neurons and reprogram glutamine metabolism to enable their growth. In this review, we examine the functions of glutamate/glutamine in the brain and how brain tumor cells reprogram glutamine metabolism. Altered glutamine metabolism can be leveraged to develop non-invasive imaging strategies and we review these imaging modalities. Finally, we examine if targeting glutamine metabolism could serve as a th...

Proceedings of the National Academy of Sciences of the United States of America, Jan 29, 2016

The ability to monitor the efficacy of an anticancer treatment in real time can have a critical e... more The ability to monitor the efficacy of an anticancer treatment in real time can have a critical effect on the outcome. Currently, clinical readouts of efficacy rely on indirect or anatomic measurements, which occur over prolonged time scales postchemotherapy or postimmunotherapy and may not be concordant with the actual effect. Here we describe the biology-inspired engineering of a simple 2-in-1 reporter nanoparticle that not only delivers a cytotoxic or an immunotherapy payload to the tumor but also reports back on the efficacy in real time. The reporter nanoparticles are engineered from a novel two-staged stimuli-responsive polymeric material with an optimal ratio of an enzyme-cleavable drug or immunotherapy (effector elements) and a drug function-activatable reporter element. The spatiotemporally constrained delivery of the effector and the reporter elements in a single nanoparticle produces maximum signal enhancement due to the availability of the reporter element in the same ce...

ACS nano, Jan 29, 2016

A major limitation of immune checkpoint inhibitors is that only a small subset of patients achiev... more A major limitation of immune checkpoint inhibitors is that only a small subset of patients achieve durable clinical responses. This necessitates the development of combinatorial regimens with immunotherapy. However, some combinations, such as MEK- or PI3K-inhibitors with a PD1-PDL1 checkpoint inhibitor, are pharmacologically challenging to implement. We rationalized that such combinations can be enabled using nanoscale supramolecular targeted therapeutics, which spatially home into tumors and exert temporally sustained inhibition of the target. Here we describe two case studies where nanoscale MEK- and PI3K-targeting supramolecular therapeutics were engineered using a quantum mechanical all-atomistic simulation-based approach. The combinations of nanoscale MEK- and PI3K-targeting supramolecular therapeutics with checkpoint PDL1 and PD1 inhibitors exert enhanced antitumor outcome in melanoma and breast cancers in vivo, respectively. Additionally, the temporal sequence of administrati...

Nanomedicine: Nanotechnology, Biology and Medicine, 2016

c-Met pathway is implicated in the resistance to anti-VEGF therapy in renal cell carcinoma (RCC).... more c-Met pathway is implicated in the resistance to anti-VEGF therapy in renal cell carcinoma (RCC). However, clinical translation of therapies targeting these pathways has been limited due to dose-limiting toxicities, feedback signaling, and low intratumoral drug accumulation. Here, we developed liposomes encapsulating a multi-receptor tyrosine kinase inhibitor (XL184) to explore the possibility of improving intratumoral concentration, enhancing antitumor efficacy and reducing toxicities. The liposomes showed increased cytotoxicity than XL184, and resulted in a sustained inhibition of phosphorylation of Met, AKT and MAPK pathways in RCC cells. In a RCC tumor xenograft model, the liposomes induced sustained inhibition of tumor growth as compared to XL184, consistent with higher inhibition of kinase signaling pathways. Biodistribution studies revealed higher accumulation of the liposomes in tumor, which translated into lower toxicities. This study shows the use of liposomes for effective inhibition of multi-kinase pathways, which can potentially emerge as a new treatment for RCC.

Biosensors & bioelectronics, Jan 27, 2017

Development of resistance to chemotherapy treatments is a major challenge in the battle against c... more Development of resistance to chemotherapy treatments is a major challenge in the battle against cancer. Although a vast repertoire of chemotherapeutics is currently available for treating cancer, a technique for rapidly identifying the right drug based on the chemo-resistivity of the cancer cells is not available and it currently takes weeks to months to evaluate the response of cancer patients to a drug. A sensitive, low-cost diagnostic assay capable of rapidly evaluating the effect of a series of drugs on cancer cells can significantly change the paradigm in cancer treatment management. Integration of microfluidics and electrical sensing modality in a 3D tumour microenvironment may provide a powerful platform to tackle this issue. Here, we report a 3D microfluidic platform that could be potentially used for a real-time deterministic analysis of the success rate of a chemotherapeutic drug in less than 12h. The platform (66mm×50mm; L×W) is integrated with the microsensors (interdigi...

ChemInform, 2014

Mesoporous nanoparticles are special examples of silica nanoparticles. They are well known for th... more Mesoporous nanoparticles are special examples of silica nanoparticles. They are well known for their variety of applications in various fields including biomedicine. This has been attributed to their size, tenability and easily modifiable capabilities because of the presence of functional groups, and their biocompatibility. In this review, we specifically focus on different modes of surface modifications using amino propyl and organic chains which facilitate maximum drug loading and sustained release. This review provides complete information about the importance of surface modifications and their biological consequences that will be helpful to understand the use of MCM-41 and SBA 15 as suitable drug delivery systems. Fig. 1 Schematic representation of MCM-41 (A) and SBA-15 (B).

ACS nano, Jan 29, 2016

In the chemical world, evolution is mirrored in the origin of nanoscale supramolecular structures... more In the chemical world, evolution is mirrored in the origin of nanoscale supramolecular structures from molecular subunits. The complexity of function acquired in a supramolecular system over a molecular subunit can be harnessed in the treatment of cancer. However, the design of supramolecular nanostructures is hindered by a limited atomistic level understanding of interactions between building blocks. Here, we report the development of a computational algorithm, which we term Volvox after the first multicellular organism, that sequentially integrates quantum mechanical energy-state- and force-field-based models with large-scale all-atomistic explicit water molecular dynamics simulations to design stable nanoscale lipidic supramolecular structures. In one example, we demonstrate that Volvox enables the design of a nanoscale taxane supramolecular therapeutic. In another example, we demonstrate that Volvox can be extended to optimizing the ratio of excipients to form a stable nanoscale...

ACS Nano, 2016

The development of resistance is the major cause of mortality in cancer. Combination chemotherapy... more The development of resistance is the major cause of mortality in cancer. Combination chemotherapy is used clinically to reduce the probability of evolution of resistance. A similar trend toward the use of combinations of drugs is also emerging in the application of cancer nanomedicine. However, should a combination of two drugs be delivered from a single nanoparticle or should they be delivered in two different nanoparticles for maximal efficacy? We explored these questions in the context of adaptive resistance, which emerges as a phenotypic response of cancer cells to chemotherapy. We studied the phenotypic dynamics of breast cancer cells under cytotoxic chemotherapeutic stress and analyzed the data using a phenomenological mathematical model. We demonstrate that cancer cells can develop adaptive resistance by entering into a predetermined transitional trajectory that leads to phenocopies of inherently chemoresistant cancer cells. Disrupting this deterministic program requires a unique combination of inhibitors and cytotoxic agents. Using two such combinations, we demonstrate that a 2-in-1 nanomedicine can induce greater antitumor efficacy by ensuring that the origins of adaptive resistance are terminated by deterministic spatially constrained delivery of both drugs to the target cells. In contrast, a combination of free-form drugs or two nanoparticles, each carrying a single payload, is less effective, arising from a stochastic distribution to cells. These findings suggest that 2-in-1 nanomedicines could emerge as an important strategy for targeting adaptive resistance, resulting in increased antitumor efficacy.

Cancer Research

Ependymomas are fatal brain malignancies with very few treatment options. More than 70% of suprat... more Ependymomas are fatal brain malignancies with very few treatment options. More than 70% of supratentorial (ST) ependymomas harbor fusions of the zinc-finger containing, chromatin modifier ZFTA and the transcriptional activator of NF-κB signaling, RELA. Oncogene-driven metabolic reprogramming is a fundamental hallmark of cancer that enables sustained tumor proliferation. ZFTA-RELA fusion protein is essential for tumorigenesis and our goal is to determine how it drives metabolism in ST-ependymomas. To address this, we developed an in vitro isogenic system by expressing the ZFTA-RELA fusion protein in immortalized mouse neural stem cells (ZFTA-RELAFUS). Using this system, we show that these tumor cells selectively upregulate expression of the glutamine transporter (SLC1A5), glutaminase (GLS), and many downstream enzymes in the glutamine metabolic pathway. Therefore, we hypothesized that the ZFTA-RELA fusion drives glutamine metabolism in ST-ependymomas. We further demonstrate that ZFTA...

Science Translational Medicine

Description Childhood posterior fossa group A ependymomas demonstrate integrated metabolic and ep... more Description Childhood posterior fossa group A ependymomas demonstrate integrated metabolic and epigenetic pathways that can be disrupted by metformin. Understanding childhood ependymomas Children with posterior fossa group A (PFA) ependymomas have a dismal prognosis. PFAs overexpress the protein EZHIP (EZH inhibitory protein), and Panwalkar and colleagues investigated whether EZHIP could epigenetically rewire metabolic pathways. Glycolysis and tricarboxylic acid (TCA) cycle metabolism were enhanced in tumors with high EZHIP expression, associated with enrichment of histone H3 lysine 27 acetylation at hexokinase-2, pyruvate dehydrogenase, and AMPKα-2. The antidiabetic drug metformin, an AMPK activator, altered histone marks, lowered EZHIP concentrations, and suppressed TCA cycle metabolism, with decreased tumor volumes and increased survival in mice carrying patient-derived xenografts. These findings suggest that targeting integrated epigenetic and metabolomic pathways could be a therapeutic strategy for children with PFAs. Childhood posterior fossa group A ependymomas (PFAs) have limited treatment options and bear dismal prognoses compared to group B ependymomas (PFBs). PFAs overexpress the oncohistone-like protein EZHIP (enhancer of Zeste homologs inhibitory protein), causing global reduction of repressive histone H3 lysine 27 trimethylation (H3K27me3), similar to the oncohistone H3K27M. Integrated metabolic analyses in patient-derived cells and tumors, single-cell RNA sequencing of tumors, and noninvasive metabolic imaging in patients demonstrated enhanced glycolysis and tricarboxylic acid (TCA) cycle metabolism in PFAs. Furthermore, high glycolytic gene expression in PFAs was associated with a poor outcome. PFAs demonstrated high EZHIP expression associated with poor prognosis and elevated activating mark histone H3 lysine 27 acetylation (H3K27ac). Genomic H3K27ac was enriched in PFAs at key glycolytic and TCA cycle–related genes including hexokinase-2 and pyruvate dehydrogenase. Similarly, mouse neuronal stem cells (NSCs) expressing wild-type EZHIP (EZHIP-WT) versus catalytically attenuated EZHIP-M406K demonstrated H3K27ac enrichment at hexokinase-2 and pyruvate dehydrogenase, accompanied by enhanced glycolysis and TCA cycle metabolism. AMPKα-2, a key component of the metabolic regulator AMP-activated protein kinase (AMPK), also showed H3K27ac enrichment in PFAs and EZHIP-WT NSCs. The AMPK activator metformin lowered EZHIP protein concentrations, increased H3K27me3, suppressed TCA cycle metabolism, and showed therapeutic efficacy in vitro and in vivo in patient-derived PFA xenografts in mice. Our data indicate that PFAs and EZHIP-WT–expressing NSCs are characterized by enhanced glycolysis and TCA cycle metabolism. Repurposing the antidiabetic drug metformin lowered pathogenic EZHIP, increased H3K27me3, and suppressed tumor growth, suggesting that targeting integrated metabolic/epigenetic pathways is a potential therapeutic strategy for treating childhood ependymomas.

Science Translational Medicine

Description Histone H3.3G34R/V mutant gliomas demonstrate LIF/STAT pathway activation that can be... more Description Histone H3.3G34R/V mutant gliomas demonstrate LIF/STAT pathway activation that can be disrupted for therapeutic benefit. STAT3 inhibition for H3.3G34R/V gliomas High-grade gliomas with arginine or valine substitutions at the glycine-34 residue on histone H3.3 (H3.3G34R/V gliomas) carry a poor prognosis. Here, Sweha and colleagues investigated the epigenetics of these tumors, finding activating epigenetic modifications on histone H3 lysine residues, as well as DNA promoter hypomethylation. They also found redistribution of repressive histone marks at the leukemia inhibitory factor (LIF) locus, leading to increased LIF abundance and secretion. LIF activated STAT3 signaling, which the authors targeted with a blood-brain barrier–permeable small-molecule inhibitor called WP1066. When given to mice carrying H3.3G34R/V tumors, WP1066 suppressed tumor growth, suggesting that inhibition of STAT3 signaling may be of therapeutic value in H3.3G34R/V gliomas. High-grade gliomas with arginine or valine substitutions of the histone H3.3 glycine-34 residue (H3.3G34R/V) carry a dismal prognosis, and current treatments, including radiotherapy and chemotherapy, are not curative. Because H3.3G34R/V mutations reprogram epigenetic modifications, we undertook a comprehensive epigenetic approach using ChIP sequencing and ChromHMM computational analysis to define therapeutic dependencies in H3.3G34R/V gliomas. Our analyses revealed a convergence of epigenetic alterations, including (i) activating epigenetic modifications on histone H3 lysine (K) residues such as H3K36 trimethylation (H3K36me3), H3K27 acetylation (H3K27ac), and H3K4 trimethylation (H3K4me3); (ii) DNA promoter hypomethylation; and (iii) redistribution of repressive histone H3K27 trimethylation (H3K27me3) to intergenic regions at the leukemia inhibitory factor (LIF) locus to drive increased LIF abundance and secretion by H3.3G34R/V cells. LIF activated signal transducer and activator of transcription 3 (STAT3) signaling in an autocrine/paracrine manner to promote survival of H3.3G34R/V glioma cells. Moreover, immunohistochemistry and single-cell RNA sequencing from H3.3G34R/V patient tumors revealed high STAT3 protein and RNA expression, respectively, in tumor cells with both inter- and intratumor heterogeneity. We targeted STAT3 using a blood-brain barrier–penetrable small-molecule inhibitor, WP1066, currently in clinical trials for adult gliomas. WP1066 treatment resulted in H3.3G34R/V tumor cell toxicity in vitro and tumor suppression in preclinical mouse models established with KNS42 cells, SJ-HGGx42-c cells, or in utero electroporation techniques. Our studies identify the LIF/STAT3 pathway as a key epigenetically driven and druggable vulnerability in H3.3G34R/V gliomas. This finding could inform development of targeted, combination therapies for these lethal brain tumors.

Cancer Research

This study uncovers a molecular mechanism linking drug-induced resistance and tumor immunity and ... more This study uncovers a molecular mechanism linking drug-induced resistance and tumor immunity and provides novel engineered solutions that target these mechanisms in the tumor and improve immunity, thus mitigating off-target effects. Drug-induced resistance, or tolerance, is an emerging yet poorly understood failure of anticancer therapy. The interplay between drug-tolerant cancer cells and innate immunity within the tumor, the consequence on tumor growth, and therapeutic strategies to address these challenges remain undescribed. Here, we elucidate the role of taxane-induced resistance on natural killer (NK) cell tumor immunity in triple-negative breast cancer (TNBC) and the design of spatiotemporally controlled nanomedicines, which boost therapeutic efficacy and invigorate “disabled” NK cells. Drug tolerance limited NK cell immune surveillance via drug-induced depletion of the NK-activating ligand receptor axis, NK group 2 member D, and MHC class I polypeptide-related sequence A, B. Systems biology supported by empirical evidence revealed the heat shock protein 90 (Hsp90) simultaneously controls immune surveillance and persistence of drug-treated tumor cells. On the basis of this evidence, we engineered a “chimeric” nanotherapeutic tool comprising taxanes and a cholesterol-tethered Hsp90 inhibitor, radicicol, which targets the tumor, reduces tolerance, and optimally reprimes NK cells via prolonged induction of NK-activating ligand receptors via temporal control of drug release in vitro and in vivo. A human ex vivo TNBC model confirmed the importance of NK cells in drug-induced death under pressure of clinically approved agents. These findings highlight a convergence between drug-induced resistance, the tumor immune contexture, and engineered approaches that consider the tumor and microenvironment to improve the success of combinatorial therapy. Significance: This study uncovers a molecular mechanism linking drug-induced resistance and tumor immunity and provides novel engineered solutions that target these mechanisms in the tumor and improve immunity, thus mitigating off-target effects.

Cellular and Molecular Bioengineering

Introduction-Immune checkpoint inhibitors that boost cytotoxic T cell-based immune responses have... more Introduction-Immune checkpoint inhibitors that boost cytotoxic T cell-based immune responses have emerged as one of the most promising approaches in cancer treatment. However, it is increasingly being realized that T cell activation needs to be rationally combined with molecularly targeted therapeutics for a maximal anti-tumor outcome. Currently, two oncogenic drivers, MAPK and PI3K-mTOR have emerged as the two main molecular targets for combining with immunotherapy. However, there are major challenges in enabling such combinations: first, such combinations can result in high rates of toxicity. Second, while, these molecular targets could be driving tumor progression, they are essential for activation of the immune cells. So, the kinase inhibitors and immunotherapy can antagonize each other. Objectives-We rationalized that the synergistic combination of kinase inhibitors and immunotherapy could be enabled by dual inhibitors-loaded supramolecular nanotherapeutics (DiLN) that can co-deliver PI3K-and MAPK-inhibitors to the cancer cells and activate immune response by T cellmodulating immunotherapy, resulting in greater anti-tumor efficacy while minimizing toxicity. Methods-We engineered DiLNs by designing the amphiphilic building blocks (both drugs and co-lipids) that enables supramolecular nanoassembly. DiLNs were tested for their physiochemical properties including size, morphology, stability and drug release kinetics profiles. The efficacy of DiLNs was tested in drug-resistant cells such as BRAF V600E melanoma (D4M), Clear cell ovarian carcinoma (TOV21G) cells. The tumor inhibition efficiency of DiLNs in combination with immune checkpoint inhibitor antibody was studied in syngeneic D4M animal model. Results-DiLNs were stable for over a month and released the drugs in a sustained manner. In vitro cytotoxicity studies in D4M and TOV21G cells showed that DiLNs were significantly more effective than free drugs. In vivo studies showed that the combination of DiLNs with anti PD-L1 antibody resulted in superior antitumor effect and survival. Conclusion-This study shows that the rational combination of DiLNs that target multiple oncogenic signaling pathways with immune checkpoint inhibitors could emerge as an effective strategy to improve immunotherapeutic response against drug resistant tumors.

Nature Biomedical Engineering

Effectively activating macrophages that can 'eat' cancer cells is challenging. In particular, can... more Effectively activating macrophages that can 'eat' cancer cells is challenging. In particular, cancer cells secrete macrophage colony stimulating factor (MCSF), which polarizes tumour-associated macrophages from an antitumour M1 phenotype to a pro-tumorigenic M2 phenotype. Also, cancer cells can express CD47, a 'don't eat me' signal that ligates with the signal regulatory protein alpha (SIRPα) receptor on macrophages to prevent phagocytosis. Here, we show that a supramolecular assembly consisting of amphiphiles inhibiting the colony stimulating factor 1 receptor (CSF-1R) and displaying SIRPα-blocking antibodies with a drug-to-antibody ratio of 17,000 can disable both mechanisms. The supramolecule homes onto SIRPα on macrophages, blocking the CD47-SIRPα signalling axis while sustainedly inhibiting CSF-1R. The supramolecule enhances M2-to-M1 repolarization within the tumour microenvironment, and significantly improves antitumour and antimetastatic efficacies in two aggressive animal models of melanoma and breast cancer, with respect to clinically available small-molecule and biologic inhibitors of CSF-1R signalling. Simultaneously blocking the CD47-SIRPα and MCSF-CSF-1R signalling axes may constitute a promising immunotherapy.

Acta neuropathologica, 2018

Diffuse midline gliomas harboring a recurrent H3 lysine 27-to-methionine (p.Lys27Met, H3K27M) con... more Diffuse midline gliomas harboring a recurrent H3 lysine 27-to-methionine (p.Lys27Met, H3K27M) constitute a recently defined pathologic entity with a particularly poor prognosis. They mainly occur in midline structures, such as the pons and thalamus of children and young adults, and are highly infiltrative [2]. More recently, case reports have described the H3K27M mutation in circumscribed (non-diffuse) gliomas, many of which are low-grade (e.g., pilocytic astrocytoma, ganglioglioma) [1, 3–5] (additional references are provided in Online Resource 1). Because of the rarity of these tumors, it is unknown whether they carry the poor clinical outcome ascribed to H3K27M-mutant infiltrating gliomas of the midline. Here, we address this gap in our knowledge by performing an integrated meta-analysis on collated clinical and pathologic data from published studies, data repositories, and collaborative efforts. A systematic search of the literature was performed from 2012 to November, 2017. Tumors were categorized based on diagnosis, location, histologic grade, growth pattern, and H3K27M mutation status. IDH 1/2-mutant diffuse gliomas were excluded to avoid bias arising from the good prognosis attributed to this mutation. Circumscribed gliomas included pilocytic astrocytoma, ganglioglioma/glioneuronal tumor/ ganglion cell tumor, pleomorphic xanthoastrocytoma, and ependymoma. Data from The Cancer Genome Atlas (TCGA) (http ://canc erge nome .nih.gov/) were obtained through the cBioPortal and PedcBioPortal for Cancer Genomics. Factors extracted included age, sex, overall survival, tumor location, histopathologic diagnosis, and WHO grade. Patient samples were then cross-referenced across studies to filter out duplicate data. Cases were also acquired from our inhouse sequencing efforts and through collaboration with the Mayo Clinic in Rochester, Minnesota. Co-occurring mutations were also documented. The endpoint extracted from all data sources was overall survival (OS). Survival functions were estimated using the Kaplan–Meier method and differences analyzed with the log-rank (Mantel–Cox) test using GraphPad Prism software (version 7). The Coxproportional hazards model was used to calculate hazard ratios (HR) using SPSS (version 24, IBM). All studies were approved by ethics committees at the respective institutions (see Online Resource 1 for further details of the methods). Among published studies and through institutional collaboration, we identified 28 cases of H3K27M-mutant circumscribed gliomas (grade I: n = 19; grade III, n = 9). Histopathology included pilocytic astrocytoma (n = 7), ganglioglioma (n = 10), anaplastic ganglioglioma (n = 3), glioneuronal tumor (n = 1), anaplastic glioneuronal tumor (n = 1), ganglion cell tumor (n = 1; Fig. 1a–d), anaplastic ependymoma (n = 3), and circumscribed glioma, not further specified (n = 2) (see Online Resource 2 for references and case details). Strikingly, more than 96% (n = 26/27 cases with location provided) of H3K27M-mutant circumscribed gliomas occurred within the midline, including the brainstem (n = 7), thalamus (n = 5), cerebellum (n = 2), spinal cord (n = 8), and other midline regions (peduncle, posterior fossa, and midline-not further specified; n = 4) (Fig. 1g). Of the remaining two cases, one was reported in the cerebrum, while the location was not specified for the other. H3K27M mutations occurred in H3F3A (n = 21), HIST1H3B (n = 1), Electronic supplementary material The online version of this article (http s://doi.org/10.1007 /s004 01-018-1805 -3) contains supplementary material, which is available to authorized users.

Cancer Research

Epigenetic modifications including altered DNA methylation and histone posttranslational modifica... more Epigenetic modifications including altered DNA methylation and histone posttranslational modifications (PTM) are central to the biology of several cancers. These modifications can regulate DNA accessibility and consequently, gene expression. In this issue, Wojcik and colleagues explore epigenetic drivers of malignant peripheral nerve sheath tumors (MPNST) harboring loss-of-function polycomb-repressive complex 2 mutations. They demonstrate alterations in specific histone PTMs and a global increase in DNA methylation. Notably, epigenetic alterations related with aberrant upregulation of proteins involved in immune evasion, which informed identification of potential therapeutic vulnerabilities. This study helps understand the complex biology of MPNSTs and may enable future therapeutic development.See related article by Wojcik et al., p. 3205.

Cancers

Altered metabolism is a hallmark of cancer cells. Tumor cells rewire their metabolism to support ... more Altered metabolism is a hallmark of cancer cells. Tumor cells rewire their metabolism to support their uncontrolled proliferation by taking up nutrients from the microenvironment. The amino acid glutamine is a key nutrient that fuels biosynthetic processes including ATP generation, redox homeostasis, nucleotide, protein, and lipid synthesis. Glutamine as a precursor for the neurotransmitter glutamate, and plays a critical role in the normal functioning of the brain. Brain tumors that grow in this glutamine/glutamate rich microenvironment can make synaptic connections with glutamatergic neurons and reprogram glutamine metabolism to enable their growth. In this review, we examine the functions of glutamate/glutamine in the brain and how brain tumor cells reprogram glutamine metabolism. Altered glutamine metabolism can be leveraged to develop non-invasive imaging strategies and we review these imaging modalities. Finally, we examine if targeting glutamine metabolism could serve as a th...

Proceedings of the National Academy of Sciences of the United States of America, Jan 29, 2016

The ability to monitor the efficacy of an anticancer treatment in real time can have a critical e... more The ability to monitor the efficacy of an anticancer treatment in real time can have a critical effect on the outcome. Currently, clinical readouts of efficacy rely on indirect or anatomic measurements, which occur over prolonged time scales postchemotherapy or postimmunotherapy and may not be concordant with the actual effect. Here we describe the biology-inspired engineering of a simple 2-in-1 reporter nanoparticle that not only delivers a cytotoxic or an immunotherapy payload to the tumor but also reports back on the efficacy in real time. The reporter nanoparticles are engineered from a novel two-staged stimuli-responsive polymeric material with an optimal ratio of an enzyme-cleavable drug or immunotherapy (effector elements) and a drug function-activatable reporter element. The spatiotemporally constrained delivery of the effector and the reporter elements in a single nanoparticle produces maximum signal enhancement due to the availability of the reporter element in the same ce...

ACS nano, Jan 29, 2016

A major limitation of immune checkpoint inhibitors is that only a small subset of patients achiev... more A major limitation of immune checkpoint inhibitors is that only a small subset of patients achieve durable clinical responses. This necessitates the development of combinatorial regimens with immunotherapy. However, some combinations, such as MEK- or PI3K-inhibitors with a PD1-PDL1 checkpoint inhibitor, are pharmacologically challenging to implement. We rationalized that such combinations can be enabled using nanoscale supramolecular targeted therapeutics, which spatially home into tumors and exert temporally sustained inhibition of the target. Here we describe two case studies where nanoscale MEK- and PI3K-targeting supramolecular therapeutics were engineered using a quantum mechanical all-atomistic simulation-based approach. The combinations of nanoscale MEK- and PI3K-targeting supramolecular therapeutics with checkpoint PDL1 and PD1 inhibitors exert enhanced antitumor outcome in melanoma and breast cancers in vivo, respectively. Additionally, the temporal sequence of administrati...

Nanomedicine: Nanotechnology, Biology and Medicine, 2016

c-Met pathway is implicated in the resistance to anti-VEGF therapy in renal cell carcinoma (RCC).... more c-Met pathway is implicated in the resistance to anti-VEGF therapy in renal cell carcinoma (RCC). However, clinical translation of therapies targeting these pathways has been limited due to dose-limiting toxicities, feedback signaling, and low intratumoral drug accumulation. Here, we developed liposomes encapsulating a multi-receptor tyrosine kinase inhibitor (XL184) to explore the possibility of improving intratumoral concentration, enhancing antitumor efficacy and reducing toxicities. The liposomes showed increased cytotoxicity than XL184, and resulted in a sustained inhibition of phosphorylation of Met, AKT and MAPK pathways in RCC cells. In a RCC tumor xenograft model, the liposomes induced sustained inhibition of tumor growth as compared to XL184, consistent with higher inhibition of kinase signaling pathways. Biodistribution studies revealed higher accumulation of the liposomes in tumor, which translated into lower toxicities. This study shows the use of liposomes for effective inhibition of multi-kinase pathways, which can potentially emerge as a new treatment for RCC.

Biosensors & bioelectronics, Jan 27, 2017

Development of resistance to chemotherapy treatments is a major challenge in the battle against c... more Development of resistance to chemotherapy treatments is a major challenge in the battle against cancer. Although a vast repertoire of chemotherapeutics is currently available for treating cancer, a technique for rapidly identifying the right drug based on the chemo-resistivity of the cancer cells is not available and it currently takes weeks to months to evaluate the response of cancer patients to a drug. A sensitive, low-cost diagnostic assay capable of rapidly evaluating the effect of a series of drugs on cancer cells can significantly change the paradigm in cancer treatment management. Integration of microfluidics and electrical sensing modality in a 3D tumour microenvironment may provide a powerful platform to tackle this issue. Here, we report a 3D microfluidic platform that could be potentially used for a real-time deterministic analysis of the success rate of a chemotherapeutic drug in less than 12h. The platform (66mm×50mm; L×W) is integrated with the microsensors (interdigi...

ChemInform, 2014

Mesoporous nanoparticles are special examples of silica nanoparticles. They are well known for th... more Mesoporous nanoparticles are special examples of silica nanoparticles. They are well known for their variety of applications in various fields including biomedicine. This has been attributed to their size, tenability and easily modifiable capabilities because of the presence of functional groups, and their biocompatibility. In this review, we specifically focus on different modes of surface modifications using amino propyl and organic chains which facilitate maximum drug loading and sustained release. This review provides complete information about the importance of surface modifications and their biological consequences that will be helpful to understand the use of MCM-41 and SBA 15 as suitable drug delivery systems. Fig. 1 Schematic representation of MCM-41 (A) and SBA-15 (B).

ACS nano, Jan 29, 2016

In the chemical world, evolution is mirrored in the origin of nanoscale supramolecular structures... more In the chemical world, evolution is mirrored in the origin of nanoscale supramolecular structures from molecular subunits. The complexity of function acquired in a supramolecular system over a molecular subunit can be harnessed in the treatment of cancer. However, the design of supramolecular nanostructures is hindered by a limited atomistic level understanding of interactions between building blocks. Here, we report the development of a computational algorithm, which we term Volvox after the first multicellular organism, that sequentially integrates quantum mechanical energy-state- and force-field-based models with large-scale all-atomistic explicit water molecular dynamics simulations to design stable nanoscale lipidic supramolecular structures. In one example, we demonstrate that Volvox enables the design of a nanoscale taxane supramolecular therapeutic. In another example, we demonstrate that Volvox can be extended to optimizing the ratio of excipients to form a stable nanoscale...

ACS Nano, 2016

The development of resistance is the major cause of mortality in cancer. Combination chemotherapy... more The development of resistance is the major cause of mortality in cancer. Combination chemotherapy is used clinically to reduce the probability of evolution of resistance. A similar trend toward the use of combinations of drugs is also emerging in the application of cancer nanomedicine. However, should a combination of two drugs be delivered from a single nanoparticle or should they be delivered in two different nanoparticles for maximal efficacy? We explored these questions in the context of adaptive resistance, which emerges as a phenotypic response of cancer cells to chemotherapy. We studied the phenotypic dynamics of breast cancer cells under cytotoxic chemotherapeutic stress and analyzed the data using a phenomenological mathematical model. We demonstrate that cancer cells can develop adaptive resistance by entering into a predetermined transitional trajectory that leads to phenocopies of inherently chemoresistant cancer cells. Disrupting this deterministic program requires a unique combination of inhibitors and cytotoxic agents. Using two such combinations, we demonstrate that a 2-in-1 nanomedicine can induce greater antitumor efficacy by ensuring that the origins of adaptive resistance are terminated by deterministic spatially constrained delivery of both drugs to the target cells. In contrast, a combination of free-form drugs or two nanoparticles, each carrying a single payload, is less effective, arising from a stochastic distribution to cells. These findings suggest that 2-in-1 nanomedicines could emerge as an important strategy for targeting adaptive resistance, resulting in increased antitumor efficacy.