Providing Palliative Care to Patients Throughout the State of Indiana from a Centralized Virtual Palliative Care Hub.

BACKGROUND: Access to palliative care clinicians is a limited resource. Expanding the reach of existing palliative care expertise by utilizing virtual care is one strategy to reach areas that lack access. We delivered virtual services through a centralized hub across multiple health settings and tracked outcomes. METHODS: Through a centralized virtual palliative care hub based in an urban academic health center, access to specialty palliative care was offered across homes, critical access hospitals (CAHs), and extended care facilities (ECFs) in the state of Indiana. Webpage-based platforms were used, and hardware included a cart on wheels for rural hospital sites. Data specific to palliative care were monitored for each encounter. RESULTS: Over one year, 372 patients were seen for virtual palliative care consultations, of whom 275 (73.9%) were seen in the outpatient setting (where the patient was at home or in an ECF) and 97 (26.1%) were inpatient visits performed in CAHs. Visits occurred with patients from almost all counties in Indiana. Advance directives were established for 286 (76.9%) patients seen, and 107 (28.8%) patients were referred to hospice. CONCLUSION: Specialty palliative care is a limited resource that has been further constrained by the COVID-19 pandemic. Our experience demonstrates that centralized virtual hub-based palliative care can be leveraged to provide effective, patient-centered, and compassionate care in regions without a specialist and has the potential to improve access to specialty palliative care.

Cdkn2a Loss in a Model of Neurofibroma Demonstrates Stepwise Tumor Progression to Atypical Neurofibroma and MPNST.

Plexiform neurofibromas are benign nerve sheath Schwann cell tumors characterized by biallelic mutations in the neurofibromatosis type 1 (NF1) tumor suppressor gene. Atypical neurofibromas show additional frequent loss of CDKN2A/Ink4a/Arf and may be precursor lesions of aggressive malignant peripheral nerve sheath tumors (MPNST). Here we combined loss of Nf1 in developing Schwann cells with global Ink4a/Arf loss and identified paraspinal plexiform neurofibromas and atypical neurofibromas. Upon transplantation, atypical neurofibromas generated genetically engineered mice (GEM)-PNST similar to human MPNST, and tumors showed reduced p16INK4a protein and reduced senescence markers, confirming susceptibility to transformation. Superficial GEM-PNST contained regions of nerve-associated plexiform neurofibromas or atypical neurofibromas and grew rapidly on transplantation. Transcriptome analyses showed similarities to corresponding human tumors. Thus, we recapitulated nerve tumor progression in NF1 and provided preclinical platforms for testing therapies at each tumor grade. These results support a tumor progression model in which loss of NF1 in Schwann cells drives plexiform neurofibromas formation, additional loss of Ink4a/Arf contributes to atypical neurofibromas formation, and further changes underlie transformation to MPNST. SIGNIFICANCE: New mouse models recapitulate the stepwise progression of NF1 tumors and will be useful to define effective treatments that halt tumor growth and tumor progression in NF1.

Polo-like kinase 1 as a therapeutic target for malignant peripheral nerve sheath tumors (MPNST) and schwannomas.

Neurofibromatosis type 1 (NF1) and Neurofibromatosis type 2 (NF2) are two dominantly inherited disorders that cause tumors in Schwann cells. NF1 patients have a high risk for malignant peripheral nerve sheath tumors (MPNST), which are often inoperable and do not respond well to current chemotherapies or radiation. NF2 patients have a high risk for schwannomas. To identify potential therapeutic targets in these two tumors, we screened the NF1 MPNST cell line, ST88-14, and the NF2 schwannoma cell line, HEI-193, against ~2000 drugs of known mechanisms of action (including ~600 cancer relevant drugs), and also screened the cell lines against an siRNA library targeting most protein kinases. Both the drug screen and the siRNA screen identified Polo-like kinase 1 (PLK1) among the most potent hits in both cell lines. Since PLK1 acts on the cell cycle primarily at the G2/M transition, the same stage where aurora kinase (AURKA) acts, we explored PLK1 and its relationship to aurora kinase in MPNST. Quantitative profiling of PLK1 inhibitors against a panel of 10 neurofibromatosis cell lines found that they were potent inhibitors and, unlike AURKA inhibitors, were not more selective for NF1 over NF2 tumor cells. Furthermore, one PLK1 inhibitor, BI6727 stabilized tumor volume in MPNST xenografts. We conclude that PLK1 is a therapeutic target for MPNSTs and schwannomas, but inhibitors may have a narrow therapeutic index that limits their use as a single agent.

Aurora A kinase inhibition enhances oncolytic herpes virotherapy through cytotoxic synergy and innate cellular immune modulation.

Malignant peripheral nerve sheath tumor (MPNST) and neuroblastoma models respond to the investigational small molecule Aurora A kinase inhibitor, alisertib. We previously reported that MPNST and neuroblastomas are also susceptible to oncolytic herpes virus (oHSV) therapy. Herein, we show that combination of alisertib and HSV1716, a virus derived from HSV-1 and attenuated by deletion of RL1, exhibits significantly increased antitumor efficacy compared to either monotherapy. Alisertib and HSV1716 reduced tumor growth and increased survival in two xenograft models of MPNST and neuroblastoma. We found the enhanced antitumor effect was due to multiple mechanisms that likely each contribute to the combination effect. First, oncolytic herpes virus increased the sensitivity of uninfected cells to alisertib cytotoxicity, a process we term virus-induced therapeutic adjuvant (VITA). Second, alisertib increased peak virus production and slowed virus clearance from tumors, both likely a consequence of it preventing virus-mediated increase of intratumoral NK cells. We also found that alisertib inhibited virus-induced accumulation of intratumoral myeloid derived suppressor cells, which normally are protumorigenic. Our data suggest that clinical trials of the combination of oHSV and alisertib are warranted in patients with neuroblastoma or MPNST.

Care of Patients at the End of Life: Surrogate Decision Making for Incapacitated Patients.

Competence is determined by a court of law, whereas physicians determine medical decision-making capacity (DMC). When patients lack DMC, a surrogate should be identified to make decisions. Ideally, patients will have created a durable power of attorney for health care. If a patient did not do this, state statutes specify which individuals can serve as surrogates; a current spouse typically is the first choice. Ideally, surrogates should use substituted judgment in making decisions. If this is not possible because the patient never shared end-of-life wishes with the surrogate, the surrogate can make decisions that, in the surrogate's opinion, are in the patient's best interests or that a reasonable individual would make. When no surrogate can be identified and a patient has no written advance directive, hospital ethics committees can assist with decisions, or, for some patients, a court will need to appoint a guardian. When there is a surrogate, difficulties can arise when family members disagree with the surrogate's decisions or when surrogates request treatments that, in the physician's opinion, would be futile or nonbeneficial. Hospital ethics committees may be able to assist in these situations, but appropriately conducted family meetings often resolve such difficulties.

CK2 blockade causes MPNST cell apoptosis and promotes degradation of ß-catenin.

Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that are a major cause of mortality of Neurofibromatosis type 1 (NF1) patients. MPNST patients have few therapeutic options available and only complete surgical resection can be curative. MPNST formation and survival are dependent on activated ß-catenin signaling. The goal of this study was to determine if inhibition of the CK2 enzyme can be therapeutically exploited in MPNSTs, given CK2's role in mainta ining oncogenic phenotypes including stabilization of ß-catenin. We found that CK2α is over-expressed in MPNSTs and is critical for maintaining cell survival, as the CK2 inhibitor, CX-4945 (Silmitasertib), and shRNA targeting CK2α each significantly reduce MPNST cell viability. These effects were preceded by loss of critical signaling pathways in MPNSTs, including destabilization of ß-catenin and TCF8. CX-4945 administration in vivo slowed tumor growth and extends survival time. We conclude that CK2 inhibition is a promising approach to blocking ß-catenin in MPNST cells, although combinatorial therapies may be required for maximal efficacy.

An ShRNA Screen Identifies MEIS1 as a Driver of Malignant Peripheral Nerve Sheath Tumors.

Malignant peripheral nerve sheath tumors (MPNST) are rare soft tissue sarcomas that are a major source of mortality in neurofibromatosis type 1 (NF1) patients. To identify MPNST driver genes, we performed a lentiviral short hairpin (sh) RNA screen, targeting all 130 genes up-regulated in neurofibroma and MPNSTs versus normal human nerve Schwann cells. NF1 mutant cells show activation of RAS/MAPK signaling, so a counter-screen in RAS mutant carcinoma cells was performed to exclude common RAS-pathway driven genes. We identified 7 genes specific for survival of MPSNT cells, including MEIS1. MEIS1 was frequently amplified or hypomethylated in human MPSNTs, correlating with elevated MEIS1 gene expression. In MPNST cells and in a genetically engineered mouse model, MEIS1 expression in developing nerve glial cells was necessary for MPNST growth. Mechanistically, MEIS1 drives MPNST cell growth via the transcription factor ID1, thereby suppressing expression of the cell cycle inhibitor p27(Kip) and maintaining cell survival.

Insertional Mutagenesis Identifies a STAT3/Arid1b/ß-catenin Pathway Driving Neurofibroma Initiation.

To identify genes and signaling pathways that initiate Neurofibromatosis type 1 (NF1) neurofibromas, we used unbiased insertional mutagenesis screening, mouse models, and molecular analyses. We mapped an Nf1-Stat3-Arid1b/ß-catenin pathway that becomes active in the context of Nf1 loss. Genetic deletion of Stat3 in Schwann cell progenitors (SCPs) and Schwann cells (SCs) prevents neurofibroma formation, decreasing SCP self-renewal and ß-catenin activity. ß-catenin expression rescues effects of Stat3 loss in SCPs. Importantly, P-STAT3 and ß-catenin expression correlate in human neurofibromas. Mechanistically, P-Stat3 represses Gsk3ß and the SWI/SNF gene Arid1b to increase ß-catenin. Knockdown of Arid1b or Gsk3ß in Stat3(fl/fl);Nf1(fl/fl);DhhCre SCPs rescues neurofibroma formation after in vivo transplantation. Stat3 represses Arid1b through histone modification in a Brg1-dependent manner, indicating that epigenetic modification plays a role in early tumorigenesis. Our data map a neural tumorigenesis pathway and support testing JAK/STAT and Wnt/ß-catenin pathway inhibitors in neurofibroma therapeutic trials.

Fatty acid synthase is a metabolic oncogene targetable in malignant peripheral nerve sheath tumors.

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas with minimal therapeutic opportunities. We observed that lipid droplets (LDs) accumulate in human MPNST cell lines and in primary human tumor samples. The goal of this study was to investigate the relevance of lipid metabolism to MPNST survival and as a possible therapeutic target. METHODS: Based on preliminary findings that MPNSTs accumulate LDs, we hypothesized that a deregulated lipid metabolism supports MPNST cell survival/proliferation rate. To test this, we examined respiration, role of fatty acid oxidation (FAO), and the enzyme fatty acid synthase involved in de novo fatty acid synthesis in MPNSTs using both genetic and pharmacological tools. RESULTS: We demonstrate that LDs accumulate in MPNST cell lines, primary human and mouse MPNST tumors, and neural crest cells. LDs from MPNST cells disappear on lipid deprivation, indicating that LDs can be oxidized as a source of energy. Inhibition of FAO decreased oxygen consumption and reduced MPNST survival, indicating that MPNST cells likely metabolize LDs through active FAO. FAO inhibition reduced oxygen consumption and survival even in the absence of exogenous lipids, indicating that lipids synthesized de novo can also be oxidized. Consequently, inhibition of de novo fatty acid synthesis, which is overexpressed in human MPNST cell lines, effectively reduced MPNST survival and delayed induction of tumor growth in vivo. CONCLUSION: Our results show that MPNSTs depend on lipid metabolic pathways and suggest that disrupting lipid metabolism could be a potential new strategy for the development of MPNST therapeutics.

Ras-driven transcriptome analysis identifies aurora kinase A as a potential malignant peripheral nerve sheath tumor therapeutic target.

PURPOSE: Patients with neurofibromatosis type 1 (NF1) develop malignant peripheral nerve sheath tumors (MPNST), which are often inoperable and do not respond well to current chemotherapies or radiation. The goal of this study was to use comprehensive gene expression analysis to identify novel therapeutic targets. EXPERIMENTAL DESIGN: Nerve Schwann cells and/or their precursors are the tumorigenic cell types in MPNST because of the loss of the NF1 gene, which encodes the RasGAP protein neurofibromin. Therefore, we created a transgenic mouse model, CNP-HRas12V, expressing constitutively active HRas in Schwann cells and defined a Ras-induced gene expression signature to drive a Bayesian factor regression model analysis of differentially expressed genes in mouse and human neurofibromas and MPNSTs. We tested functional significance of Aurora kinase overexpression in MPNST in vitro and in vivo using Aurora kinase short hairpin RNAs (shRNA) and compounds that inhibit Aurora kinase. RESULTS: We identified 2,000 genes with probability of linkage to nerve Ras signaling of which 339 were significantly differentially expressed in mouse and human NF1-related tumor samples relative to normal nerves, including Aurora kinase A (AURKA). AURKA was dramatically overexpressed and genomically amplified in MPNSTs but not neurofibromas. Aurora kinase shRNAs and Aurora kinase inhibitors blocked MPNST cell growth in vitro. Furthermore, an AURKA selective inhibitor, MLN8237, stabilized tumor volume and significantly increased survival of mice with MPNST xenografts. CONCLUSION: Integrative cross-species transcriptome analyses combined with preclinical testing has provided an effective method for identifying candidates for molecular-targeted therapeutics. Blocking Aurora kinases may be a viable treatment platform for MPNST.

Neurotrophin-4 regulates the survival of gustatory neurons earlier in development using a different mechanism than brain-derived neurotrophic factor.

The number of neurons in the geniculate ganglion that are available to innervate taste buds is regulated by neurotrophin-4 (NT-4) and brain-derived neurotrophic factor (BDNF). Our goal for the current study was to examine the timing and mechanism of NT-4-mediated regulation of geniculate neuron number during development. We discovered that NT-4 mutant mice lose 33% of their geniculate neuronal cells between E10.5 and E11.5. By E11.5, geniculate axons have just reached the tongue and do not yet innervate their gustatory targets; thus, NT-4 does not function as a target-derived growth factor. At E11.5, no difference was observed in proliferating cells or the rate at which cells exit the cell cycle between NT-4 mutant and wild type ganglia. Instead, there was an increase in TUNEL-labeling, indicating an increase in cell death in Ntf4(-/-) mice compared with wild types. However, activated caspase-3, which is up-regulated in the absence of BDNF, was not increased. This finding indicates that cell death initiated by NT-4-removal occurs through a different cell death pathway than BDNF-removal. We observed no additional postnatal loss of taste buds or neurons in Ntf4(-/-) mice. Thus, during early embryonic development, NT-4 produced in the ganglion and along the projection pathway inhibits cell death through an activated caspase-3 independent mechanism. Therefore, compared to BDNF, NT-4 plays distinct roles in gustatory development; differences include timing, source of neurotrophin, and mechanism of action.

Discovery of a small molecule targeting IRA2 deletion in budding yeast and neurofibromin loss in malignant peripheral nerve sheath tumor cells.

Malignant peripheral nerve sheath tumor (MPNST) is a life-threatening complication of neurofibromatosis type 1 (NF1). NF1 is caused by mutation in the gene encoding neurofibromin, a negative regulator of Ras signaling. There are no effective pharmacologic therapies for MPNST. To identify new therapeutic approaches targeting this dangerous malignancy, we developed assays in NF1(+/+) and NF1(-/-) MPNST cell lines and in budding yeast lacking the NF1 homologue IRA2 (ira2Δ). Here, we describe UC1, a small molecule that targets NF1(-/-) cell lines and ira2Δ budding yeast. By using yeast genetics, we identified NAB3 as a high-copy suppressor of UC1 sensitivity. NAB3 encodes an RNA binding protein that associates with the C-terminal domain of RNA Pol II and plays a role in the termination of nonpolyadenylated RNA transcripts. Strains with deletion of IRA2 are sensitive to genetic inactivation of NAB3, suggesting an interaction between Ras signaling and Nab3-dependent transcript termination. This work identifies a lead compound and a possible target pathway for NF1-associated MPNST, and shows a novel model system approach to identify and validate target pathways for cancer cells in which NF1 loss drives tumor formation.

Lingual and palatal gustatory afferents each depend on both BDNF and NT-4, but the dependence is greater for lingual than palatal afferents.

Neurons of the geniculate ganglion innervate taste buds located in two spatially distinct targets, the tongue and palate. About 50% of these neurons die in Bdnf(-/-) mice and Ntf4/5(-/-) mice. Bdnf(-/-)/Ntf4/5(-/-) double mutants lose 90-95% of geniculate ganglion neurons. To determine whether different subpopulations are differentially influenced by neurotrophins, we quantified neurons from two ganglion subpopulations separately and remaining taste buds at birth within each target field in wild-type, Bdnf(-/-), Ntf4/5(-/-), and Bdnf(-/-)/Ntf4/5(-/-) mice. In wild-type mice the same number of neurons innervated the anterior tongue and soft palate and each target contained the same number of taste buds. Compared to wild-type mice, Bdnf(-/-) mice showed a 50% reduction in geniculate neurons innervating the tongue and a 28% loss in neurons innervating the soft palate. Ntf4/5(-/-) mice lost 58% of the neurons innervating the tongue and 41% of the neurons innervating the soft palate. Taste bud loss was not as profound in the NT-4 null mice compared to BDNF-null mice. Tongues of Bdnf(-/-)/Ntf4/5(-/-) mice were innervated by 0 to 4 gustatory neurons and contained 3 to 16 taste buds at birth, indicating that some taste buds remain even when all innervation is lost. Thus, gustatory neurons are equally dependent on BDNF and NT-4 expression for survival, regardless of what peripheral target they innervate. However, taste buds are more sensitive to BDNF than NT-4 removal.

BDNF is required for the survival of differentiated geniculate ganglion neurons.

In mice lacking functional brain-derived neurotrophic factor (BDNF), the number of geniculate ganglion neurons, which innervate taste buds, is reduced by one-half. Here, we determined how and when BDNF regulates the number of neurons in the developing geniculate ganglion. The loss of geniculate neurons begins at embryonic day 13.5 (E13.5) and continues until E18.5 in BDNF-null mice. Neuronal loss in BDNF-null mice was prevented by the removal of the pro-apoptotic gene Bax. Thus, BDNF regulates embryonic geniculate neuronal number by preventing cell death rather than promoting cell proliferation. The number of neurofilament positive neurons expressing activated caspase-3 increased on E13.5 in bdnf(-/-) mice, compared to wild-type mice, demonstrating that differentiated neurons were dying. The axons of geniculate neurons approach their target cells, the fungiform papillae, beginning on E13.5, at which time we found robust BDNF(LacZ) expression in these targets. Altogether, our findings establish that BDNF produced in peripheral target cells regulates the survival of early geniculate neurons by inhibiting cell death of differentiated neurons on E13.5 of development. Thus, BDNF acts as a classic target-derived growth factor in the developing taste system.