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Diffuse intrinsic pontine glioma (DIPG) is the most common and deadliest pediatric brainstem tumor and is difficult to treat with chemotherapy in part due to the blood-brain barrier (BBB). Focused ultrasound (FUS) and microbubbles (MBs) have been shown to cause BBB disruption (BBBD), allowing larger chemotherapeutics to enter the parenchyma. Panobinostat is an example of a promisingin vitroagent in DIPG with poor clinical efficacy due to low BBB penetrance. In this study, we hypothesized that using FUS to disrupt the BBB allows higher concentrations of panobinostat to accumulate in the tumor, providing a therapeutic effect. Mice were orthotopically injected with a patient-derived DMG cell line, BT-245. MRI was used to guide FUS/MB (1.5 MHz, 0.615 MPa PNP, 1 Hz PRF, 10 ms PL, 3 min treatment time) / (25 µL/kg, IV) targeting to the tumor location. In animals receiving panobinostat (10 mg/kg, IP) in combination with FUS/MB, a 3-fold increase in tumor panobinostat concentration was observed, with only insignificant increase of the drug in the forebrain. In mice receiving three weekly treatments, the combination of panobinostat and FUS/MB led to a 71% reduction of tumor volumes by MRI (p= 0.01). Furthermore, FUS/MB improved the mean survival from 21 to 31 days (p< 0.0001). Our study demonstrates that FUS-mediated BBBD can increase the delivery of panobinostat to an orthotopic DMG tumor, providing a strong therapeutic effect and increased survival.One Sentence SummaryFUS and microbubbles can increase the delivery of panobinostat to a patient-derived xenograft (PDX) orthotopic DMG tumor, providing a strong therapeutic effect and increased survival.

Atypical teratoid rhabdoid tumor (ATRT) is a highly aggressive pediatric brain tumor. Despite radiation, aggressive chemotherapy and autologous stem cell rescue, children usually have a poor survival time. In the present study, the role of TP53/MDM2 interaction in ATRT was investigated. A functional genomic screen identified the TP53/MDM2 axis as a therapeutic target in the central nervous system (CNS) ATRT. Gene expression analysis revealed that all ATRT sub‑groups expressed high levels of MDM2, which is a negative regulator of TP53. Using cell viability, colony formation and methylcellulose assays it was found that genetic MDM2 inhibition with short hairpin RNA or chemical MDM2 inhibition with small molecule inhibitors, Nutlin3 and idasanutlin (RG7388) decreased the growth of ATRT cell lines. Furthermore, idasanutlin significantly decreased the growth of intracranial orthotopic ATRT brain tumors, as evaluated using T2 MRI, and prolonged survival time relative to control animals. MRI of intracranial tumors showed that diffusion coefficient, an effective marker for successful treatment, significantly increased with idasanutlin treatment showing tumor necrosis/apoptosis. Immunohistochemistry revealed an increased number of caspase‑3‑positive cells in the idasanutlin treatment group, confirming the induction of apoptosis

In a screen of over 100 FDA approved drugs on PFA 1q+ EPN cells, 5-fluorouracil (5FU) and All-Trans-Retinoic Acid (ATRA) were identified as inhibitors of EPN cell line growth. We performed in-vitro cell growth assays combining increasing doses of radiation and 5FU and found a significant synergistic effect on cell growth and apoptosis in 1q+ PFA EPN cell lines. Further growth attenuation was seen when ATRA was added 48 hours following radiation and 5FU treatment. This led us to development of preclinical studies in the 1q+ PFA orthotopic xenograft models MAF-811_XF and MAF-928_XF. In the initial cohort, tumors were allowed to establish prior to treatment start confirmed by MRI. In both MAF-811 and MAF-928, chemotherapy improved survival compared to no treatment. As consistent with standard of care, radiation significantly improved survival (p=0.0016) but there was no added benefit to combining 5FU or 5FU+ATRA with radiation. A second cohort was treated using the same treatment approach, however radiation and 5FU were started with minimal to no visible tumors by MRI. Interestingly, we found a significant increase in survival between vehicle control and combination 5FU+ATRA (HR 5.121, 95% CI: 0.2506, 2.409, p=0.048) in MAF-811 mice. However, again with radiation, there was no significant change in survival with only a single cycle of 5FU+ATRA. This led to continued maintenance of 5FU+ATRA cycles of 6 weeks with 2 weeks off for 4 cycles post radiation in mice with minimal tumor. When 5FU with radiation is followed by 5FU+ATRA and is continued in mice with minimal disease, survival significantly improved when compared to radiation alone (HR 9.020, 95% CI: 1.933 to 42.09, p=0.007). These studies highlight the importance of chemotherapy in minimal disease and is the rationale for a Phase I/II study in relapsed PFA EPN and in upfront 1q+ PFA EPN.

BACKGROUND: Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. PTEFb-mediated promoter proximal pause-release of Pol II is a conserved regulatory mechanism for synchronous transcriptional induction best described in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. DESIGN/METHOD: In order to examine the dynamics of chromatin reorganization following radiotherapy, we performed a combination of ChIP-, ATAC-, and RNA-seq in model systems of diffuse intrinsic pontine glioma (DIPG) and other pediatric high-grade gliomas (pHGG) following IR exposure. We interrogated IR-induced gene expression in the presence or absence of PTEFb blockade, including both mechanistic and functional consequences of concurrent inhibition or genetic depletion. We utilized culture models with live cell imaging to assess the therapeutic synergy of PTEFb inhibition with IR, as well as the therapeutic index of this intervention relative to normal controls. Finally, we employed orthotopic models of pHGG treated with conformal radiotherapy and CNS-penetrant PTEFb inhibitors in order to assess tolerability and anti-tumor effect in vivo. RESULTS: Rapid genome-wide redistribution of active chromatin features and PTEFb facilitates Pol II pause-release to drive nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of PTEFb imparts a transcription elongation defect, abrogating canonical adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. CONCLUSION: These studies reveal a central role for PTEFb underpinning the early adaptive response to radiotherapy, opening new avenues for combinatorial treatment in these lethal malignancies.

BACKGROUND: Though long recognized as a master regulator of cell proliferation across a wide range of cancers, Myc has proven elusive to direct therapeutic targeting. The CDK9-containing PTEFb, complexed with either BRD4 or SEC, facilitates Myc-driven transcriptional programs and is necessary for sustaining expression of Myc itself. Advances in development of clinical-grade CDK9 inhibitors creates an opportunity to examine this as a rational therapy for Myc-driven medulloblastoma. METHODS: We used both RNAi depletion and a panel of pharmacologic agents to characterize the mechanistic and functional consequences of CDK9 inhibition in Myc-driven medulloblastoma. We used a combination of clonogenic assays and live cell imaging to assess the cytotoxic effects of CDK9 activity loss. We then performed a combination of CUT&RUN and RNA-seq to evaluate alterations to Myc binding and downstream Myc-driven transcriptional programs. Finally, we employed orthotopic xenograft models of medulloblastoma to assess CNS penetration, tolerability, and anti-tumor efficacy of lead CDK9i candidate compounds. RESULTS: Genetic or pharmacologic inhibition of CDK9 leads to a loss of Myc expression and downregulation of hallmark Myc-driven transcriptional programs. This corresponds to a loss of cell fitness, as measured by decreased proliferation and clonogenic potential. Clinically relevant CDK9 inhibitors show variable efficacy in vivo, but the CNS-penetrant zotiraciclib achieved a significant prolongation in xenograft survival. CONCLUSION: CDK9 catalytic activity represents a druggable vulnerability underpinning Myc-driven transcriptional programs. The development of CNS-penetrant CDK9 inhibitors may open new avenues for rational therapy in these high-risk medulloblastomas.

Ependymoma (EPN) is an aggressive pediatric brain tumor, for which the benefits of chemotherapy in pediatric patients have not been defined. EPN treated with surgery and radiation recur in 23-66% of patients. Our group has previously established aggressive behaviors of EPN, including high tumor cellularity, cytological anaplasia, high mitotic index, tumor necrosis, and the presence of inflammatory cells such as M2-type myeloid cells. Here we report on an advanced 9.4 Tesla MRI protocol for characterizing the cellular and vascular phenotype and treatment response to chemo-radiation therapy (CRT) in an orthotopic mouse model of patient derived xenografts (PDX) of pediatric EPN . Female severely immune deficient (SCID) mice were used for intracranial inoculation of disaggregated tumors from pediatric EPN patients (n=22). High-resolution T2w-MRI was able to detect cerebellar microlesions as small as 0.2 mm diameter; the median tumor volumes at the baseline were 21±12 mm3. Using diffusion-weighted based cell-size imaging, iron-oxide based vessel-size imaging and quantitative T2-maps, the EPN-specific phenotype was characterized by an increased cell size (S=14 microns), increased vessel density index (Q=0.54), and low ADC values (0.63x10-3). Once the intracranial tumors reached at least 5 mm3, animals were treated with CRT (10 Gy radiation plus 30 mg/kg 5-fluorouracil, n=6). CRT resulted in a tumor shrinkage, tumor necrosis with decreased cell sizes and increased ADC values, and a dramatic vascular-inflammatory response (decreased Q and DT2 values with the injection of iron oxide nanoparticles as macrophage-specific contrast). In summary, orthotopically implanted PDX EPN in mice closely mimic histological features, anatomical location and radiological features of the primary tumors. A significant decrease in vessel size density and an increase in inflammatory cells were seen as soon as 2 days after CRT. The late response (2 weeks post CRT) is characterized by decreased cellularity, cell size, and tumor volumes.

Atypical Teratoid Rhabdoid Tumor is a highly aggressive pediatric brain tumor with poor prognosis driven by loss of the chromatin remodeling factor SMARCB1 that is responsible for determining cellular pluripotency and lineage commitment. The mechanisms by which SMARCB1 deletion results in tumorigenesis remain unclear. We investigated the effect of SIRT2 inhibition in ATRT which was identified as a primary dependency in ATRT. SIRT2 inhibition with shRNA or Thiomyristoyl (TM) decreased ATRT cell growth, inhibited clonogenic potential and leaded to the cell cycle arrest. SIRT2 inhibition effectively suppresses pluripotency-associated genomic programs, significantly changed stem cell frequency, decreased tumor-sphere formation of ATRT cells and attenuated tumor cell self-renewal. In vivo SIRT2 inhibition decreased oncogenic markers and increased accumulation neuronal differentiation markers. Furthermore, SIRT2 induced apoptosis, decreased tumor growth and prolonged survival in orthotopic xenograft models. Single-cell RNA transcriptome analysis of xenoftaft tumors reveals elimination of tumor cells expressing stem cell genes and expansion of tumor cells expressing differentiated genes following TM treatment in ATRT. We demonstrated that SIRT2 inhibition is a molecular vulnerability in SMARCB1-deleted ATRT.

Introduction: Identifying targets in the tumor microenvironment (TME) that act as barriers to an effective anti-tumor immune response has become an area of intense investigation. In the current study, we established EphB4-ephrin-B2 signaling as a key pathway that regulates both innate and adaptive arms of the immune system. Eph receptor tyrosine kinases and their membrane-bound ephrin ligands have been implicated in human malignancies and in immune cell development, migration, and activation in inflammatory models. However, direct evidence that supports the role of Eph-ephrin interaction in cancer-related immune response is lacking. We hypothesized that EphB4-ephrin-B2 interaction regulates TME by sustaining immunosuppressive cells-Tregs and TAMs thus negatively impacting the functional ability of CD8 T cells. Materials and methods: We used orthotopic models of head and neck squamous cell carcinoma to determine the role of EphB4-ephrin-B2 interaction in tumor immune microenvironment. Mice were treated with control agent or an EphB4-ephrin-B2 blocker in the absence or presence of radiation (RT). Tumor immune cell infiltrates were analyzed using mass cytometry and flow cytometry applications. ELISA or multiplex cytokine array were utilized to determine circulating cytokine/chemokine levels in plasma. Results: We observed that inhibition of EphB4-ephrin-B2 signaling in vivo significantly reduced tumor growth and decreased intratumoral Tregs, TAMs, and increased the activation of Teffector cells, without affecting CD4 T cell numbers. This was correlated with decreased Treg proliferation and activation when EphB4-ephrin-B2 signaling is inhibited. Since RT remains the mainstay in treatment of head and neck squamous cell cancer (HNSCC) patients, we combined EphB4-ephrin-B2 inhibitor with RT in our tumor model and observed further increase in CD8 and CD4 T cell infiltrates and activation status, and a significant decline in circulating TGF-β1 levels and an increase in CXCL10 levels compared to the control group. A significant reduction of TAMs, favoring a polarization towards an anti-tumoral M1 phenotype, was also observed in EphB4-ephrin-B2 inhibitor+RT group. We also compared the efficacy of combining EphB4-ephrin-B2 inhibitor with RT to anti-PDL1+RT in an in vivo model known to develop resistance to anti-PDL1+RT therapy. Our data demonstrated that combining EphB4-ephrin-B2 inhibitor with RT was equally effective to that of anti-PDL1+RT in terms of anti-tumor growth response. Conclusions: Our study provides the first insight into a novel role for EphB4-ephrin-B2 interaction in modulating tumor immune microenvironment in HNSCC. Our findings present a potential alternative in the form of EphB4-ephrin-B2 targeted therapeutics that can be tested in clinical trials in combination with RT for HNSCC patients. Citation Format: Shilpa Bhatia, Ayman Oweida, Shelby Lennon, Laurel Darragh, Sanjana Bukkapatnam, Benjamin Van Court, David Raben, Natalie Serkova, Antonio Jimeno, Eric Clambey, Elena Pasquale, Sana Karam. Blockade of EphB4-ephrin-B2 interaction remodels the tumor immune microenvironment in head and neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5035.

Severe ischemia/reperfusion (IR) injury is a risk factor for delayed graft function. Delayed graft function remains difficult to predict, and it currently relies primarily on serum creatinine (SCr), urine output, and occasionally on graft biopsy. (1)H-NMR (nuclear magnetic resonance spectroscopy) based metabolomics was used to establish IR-specific metabolic markers in both blood and kidney tissue. These markers were compared to SCr and graft histology.Male Lewis rats were used for kidney transplantation. Two cold ischemia (CI) groups (24- and 42-hour) and two transplantation groups [after 24 (TX24) and after 42 hours (TX42) of CI] were compared to a control group. Whole blood and kidney tissue were collected for further analysis.SCr levels taken 24 hours after transplantation were 1.6 +/- 0.12 mg/dL (TX24) and 2.1 +/- 0.5 mg/dL (TX42), (P= n.s.). Histology samples revealed mild injury in the TX24 group and severe injury in the TX42 group. A significantly decreased level of polyunsaturated fatty acids (PUFA) and elevated levels of allantoin, a marker of oxidative stress, was found in the renal tissue. In the blood, both trimethylamine-N-oxide (TMAO), a marker of renal medullary injury, and allantoin were significantly increased. Allantoin levels were low in both the control and CI groups. Levels were significantly increased after reperfusion (control 0.02 +/- 0.03 micromol/mL, TX24 1.13 +/- 0.22, and TX42 1.89 +/- 0.38, P0.001), and correlated with cold ischemia time (r= 0.96) and TMAO (r= 0.94).The (1)H-NMR metabolic profiles of both the mild and severe IR groups revealed significant changes consistent with graft histology, while the SCr did not.

Co-administration of the calcineurin inhibitor cyclosporine and the mTOR inhibitors sirolimus or everolimus increases the efficacy of immunosuppression after organ transplantation. However, clinical studies showed enhancement of cyclosporine toxicity. To characterize the biochemical mechanisms involved, we assessed the time-dependent effects of cyclosporine in combination with mTOR inhibitors on energy production (ex vivo (31)P-MRS), glucose metabolism (ex vivo (13)C-MRS), and reactive oxygen species (ROS) formation (using the fluorescent agent 2',7'-dichlorofluorescein diacetate) in perfused rat brain slices. Cyclosporine alone inhibited energy production (ATP: 75+/-9%), the Krebs cycle (4-(13)C-glutamate from 1-(13)C-glucose: 61+/-27%), and oxidative phosphorylation (NAD(+): 62+/-25%) after 4 h of perfusion. After 10 h, activation of anaerobic glycolysis (3-(13)C-lactate: 140+/-17%) compensated for inhibition of mitochondrial energy production and lowered the intracellular pH. ROS formation was increased after 4 h (285+/-55% of untreated control), but not after 10 h. mTOR inhibitors alone inhibited lactate production. When combined with cyclosporine, sirolimus enhanced cyclosporine-induced inhibition of energy metabolism (ATP: 64+/-9%) and ROS formation (367+/-46%). Most importantly, sirolimus inhibited cytosolic glycolysis and therefore compensation for cyclosporine-induced ATP reduction after 10 h. In contrast to sirolimus, everolimus antagonized cyclosporine-induced inhibition of mitochondrial energy metabolism (ATP: 91+/-7%) and ROS formation (170+/-49%). The antioxidant tocopherol antagonized all cyclosporine effects on cell metabolism. Cyclosporine time-dependently inhibited mitochondrial metabolism and increased ROS, followed by compensation involving anaerobic glycolysis. Everolimus antagonized cyclosporine-induced mitochondrial dysfunction, whereas sirolimus inhibited compensatory anaerobic glycolysis, thus enhancing cyclosporine's negative effects. ROS play the key role in mediating the negative effects of cyclosporine on cell energy metabolism.

For over two decades, calcineurin inhibitors (CIs) have been the mainstay of immunosuppressive therapy following solid-organ transplantation. However, CI nephrotoxicity is one of the main contributors to chronic kidney allograft dysfunction. A novel class of immunosuppressants that inhibit the kinase mammalian target of rapamycin (mTOR), although not nephrotoxic themselves, enhance CI nephrotoxicity. The biochemical basis of CI toxicity and their toxicodynamic interaction with mTOR inhibitors is still poorly understood. Studies using a magnetic resonance spectroscopy-based metabonomic approach indicate that CI toxicity is caused by drug-induced mitochondrial dysfunction and that mTOR inhibitors enhance the negative effects of CIs on cell energy metabolism.

Current paradigms attribute the association between aging and cancers to the accumulation of oncogenic mutations. Alternatively, we hypothesize that aging-associated reductions in the fitness of stem and progenitor cell populations should be conducive for increased selection of adaptive oncogenic mutations which can partially restore fitness, thereby promoting the initiation of cancers. We have explored how aging affects B-cell progenitor fitness and subsequently leukemogenesis. Here we show that IL-7 receptor (IL-7R) signaling is defective in aged B-cell progenitors leading to reduced phospho-STAT5 and Akt levels. Aged B-cell progenitors also exhibit altered expression of IL-7R regulated genes, including reduced Pax5, Myc and HPRT, and increased TNF-α. Both decreasing IL-7R signaling and increasing inflammation phenocopied aging-associated B-lymphopoiesis defects in young mice. Decreasing IL-7R signaling also promoted selection for Bcr-Abl expressing B-cell progenitors coinciding with increased leukemogenesis in vivo. Importantly, Bcr-Abl restored defective IL-7R signaling and inflammation-induced defects in these cells. These studies elucidate how reduced IL-7R signaling and increased inflammation promote aging-associated reductions in B-lymphopoiesis. Furthermore, we show that the selection for cells harboring oncogenic mutations (such as Bcr-Abl) is increased in aged backgrounds due in part to their ability to correct aging-associated fitness defects.

The accumulation of oncogenic mutations over time is thought drive increased cancer incidence with age. Alternatively, we hypothesize that aging reduces cellular fitness in stem and progenitor pools, leading to increased selection for oncogenic mutations and increased cancers. We have explored how aging affects the fitness of B-progenitor cells and leukemogenesis. The fitness of B-progenitors declines with age, as defined by competitive transplantation assays. Analyses of aged B-progenitors revealed increased expression of inflammatory genes, and decreased expression of genes involved in B-lineage determination, B-cell signaling, nucleotide synthesis, and other metabolic pathways. These changes correlated with decreased interleukin-7 receptor (IL-7R) mediated signaling, reduced production of ATP and other nucleotides, and perturbations in the TCA cycle in aged B-progenitors. Decreasing IL-7R signaling and inducing inflammation in young mice impaired B-lymphopoiesis, accompanied by aging-like gene expression profiles. Importantly, neutralization of IL-7 significantly increased selection for the Bcr-Abl oncogene in B-progenitor pools, coinciding with restored signaling. Similarly, Bcr-Abl is adaptive within B-progenitor pools from aged but not young mice. These studies reveal roles for IL-7R signaling and inflammation in declining B-lymphopoiesis in old age, and demonstrate how the selection for Bcr-Abl in aged lymphopoietic backgrounds can drive leukemogenesis.