Ella W. Englander

Sequencing data from different types of cancers including melanomas demonstrate that tumors with high mutational loads are more likely to respond to immune checkpoint blockade (ICB) therapies. We have previously shown that low-dose intratumoral injection of the chemotherapeutic DNA damaging drug cisplatin activates intrinsic mutagenic DNA damage tolerance pathway, and when combined with ICB regimen leads to tumor regression in the mouse YUMM1.7 melanoma model. We now report that tumors generated with anin vitrocisplatin-mutagenized YUMM1.7 clone (YUMM1.7-CM) regress in response to ICB, while an identical ICB regimen alone fails to suppress growth of tumors generated with the parental YUMM1.7 cells. Regressing YUMM1.7-CM tumors show greater infiltration of CD8 T lymphocytes, higher granzyme B expression, and higher tumoral cell death. Similarly,ex-vivo, immune cells isolated from YUMM1.7-CM tumors-draining lymph nodes (TDLNs) co-incubated with cultured YUMM1.7-CM cells, eliminate the tumor cells more efficiently than immune cells isolated from TDLNs of YUMM1.7 tumor-bearing mice. Collectively, our findings show thatin vitroinduced cisplatin mutations potentiate the antitumor immune response and ICB efficacy, akin to tumor regression achieved in the parental YUMM1.7 model by ICB administered in conjunction with intratumoral cisplatin injection. Hence, our data uphold the role of tumoral mutation burden in improving immune surveillance and response to ICB, suggesting a path for expanding the range of patients benefiting from ICB therapy.

Major advances in cancer therapy rely on engagement of the patient’s immune system and suppression of mechanisms that impede the antitumor immune response. Among the most notable is immune checkpoint blockade (ICB) therapy that releases immune cells from suppression. Although ICB has had significant success particularly in melanoma, it eradicates tumors in subsets of patients and sequencing data across different cancers suggest that tumors with high mutational loads are more likely to respond to ICB. This is consistent with the premise that greater tumoral mutational loads contribute to formation of neoantigens that spur the body’s antitumor immune response. Prompted by strong evidence supporting the therapeutic benefits of neoantigens in the context of ICB, we have developed a mouse melanoma combination treatment, where intratumoral administration of DNA damaging drug transiently activates intrinsic mutagenic DNA damage tolerance pathway and improves success rates of ICB. Using the YUMM1.7 cells melanoma model, we demonstrate that intratumoral delivery of cisplatin activates translesion synthesis DNA polymerases‐catalyzed DNA synthesis on damaged DNA, which when coupled with ICB regimen, elicits durable tumor regression. We expect that this new combination protocol affords insights with clinical relevance that will help expand the range of patients who benefit from ICB therapy.

The hypoxic environment within solid tumors impedes the efficacy of chemotherapeutic treatments. Here, we demonstrate that hypoxia augments the capacity of melanoma cells to withstand cisplatin and doxorubicin cytotoxicity. We show that B16F10 cells derived from spontaneously formed melanoma and YUMM1.7 cells, engineered to recapitulate human‐relevant melanoma driver mutations, profoundly differ in their vulnerabilities to cisplatin and doxorubicin. The differences are manifested in magnitude of proliferative arrest and cell death rates, extent of mtDNA depletion, and impairment of mitochondrial respiration. In both models, cytotoxicity is mitigated by hypoxia, which augments glycolytic metabolism. Collectively, the findings implicate metabolic reprogramming in drug evasion and suggest that melanoma tumors with distinct genetic makeup may have differential drug vulnerabilities, highlighting the importance of precision anticancer treatments.

Acute inhalation of combustion smoke produces long-term neurologic deficits in survivors. To study the mechanisms that contribute to the development of neurologic deficits and identify targets for prevention, we developed a mouse model of acute inhalation of combustion smoke, which supports longitudinal investigation of mechanisms that underlie the smoke induced inimical sequelae in the brain. Using a transgenic mouse engineered to overexpress neuroglobin, a neuroprotective oxygen-binding globin protein, we previously demonstrated that elevated neuroglobin preserves mitochondrial respiration and attenuates formation of oxidative DNA damage in the mouse brain after smoke exposure. In the current study, we show that elevated neuronal neuroglobin attenuates the persistent inflammatory changes induced by smoke exposure in the mouse brain and mitigates concordant smoke-induced long-term neurobehavioral deficits. Specifically, we found that increases in hippocampal density of GFAP and Iba-1 positive cells that are detected post-smoke in wild-type mice are absent in the neuroglobin overexpressing transgenic (Ngb-tg) mice. Similarly, the smoke induced hippocampal myelin depletion is not observed in the Ngb-tg mice. Importantly, elevated neuroglobin alleviates behavioral and memory deficits that develop after acute smoke inhalation in the wild-type mice. Taken together, our findings suggest that the protective effects exerted by neuroglobin in the brains of smoke exposed mice afford protection from long-term neurologic sequelae of acute inhalation of combustion smoke. Our transgenic mouse provides a tool for assessing the potential of elevated neuroglobin as possible strategy for management of smoke inhalation injury.

Context: Acute inhalation of combustion smoke triggers neurologic sequelae in survivors. Due to the challenges posed by heterogeneity of smoke exposures in humans, mechanistic links between acute smoke inhalation and neuropathologic sequelae have not been systematically investigated. Methods: Here, using mouse model of acute inhalation of combustion smoke, we studied longitudinal neurobehavioral manifestations of smoke exposures and molecular/cellular changes in the mouse brain. Results: Immunohistochemical analyses at eight months post-smoke, revealed hippocampal astrogliosis and microgliosis accompanied by reduced myelination. Elevated expression of proinflammatory cytokines was also detected. Longitudinal testing in different neurobehavioral paradigms in the course of post-smoke recovery, revealed lasting anxiety-like behavior. The examined paradigms included the open field exploration/anxiety testing at two, four and six months post-smoke, which detected decreases in total distance traveled and time spent in the central arena in the smoke-exposed compared to sham-control mice, suggestive of dampened exploratory activity and increased anxiety-like behavior. In agreement with reduced open field activity, cued fear conditioning test revealed increased freezing in response to conditioned auditory stimulus in mice after acute smoke inhalation. Similarly, elevated plus maze testing demonstrated lesser presence in open arms of the maze, consistent with anxiety-like behavior, for the post-smoke exposure mice. Conclusions: Taken together, our data demonstrate for the first time persistent neurobehavioral manifestations of acute inhalation of combustion smoke and provide new insights into long-term progression of events initiated by disrupted brain oxygenation that might contribute to lasting adverse sequelae in survivors of smoke inhalation injuries.

In the peripheral nervous system (PNS) in the absence of tight blood barrier, neurons are at increased risk of DNA damage, yet the question of how effectively PNS neurons manage DNA damage remains largely unanswered. Genotoxins in systemic circulation include chemotherapeutic drugs that reach peripheral neurons and damage their DNA. Because neurotoxicity of platinum-based class of chemotherapeutic drugs has been implicated in PNS neuropathies, we utilized an in vitro model of Dorsal Root Ganglia (DRGs) to investigate how peripheral neurons respond to cisplatin that forms intra- and interstrand crosslinks with their DNA. Our data revealed strong transcriptional upregulation of the translesion synthesis DNA polymerase kappa (Pol κ), while expression of other DNA polymerases remained unchanged. DNA Pol κ is involved in bypass synthesis of diverse DNA lesions and considered a vital player in cellular survival under injurious conditions. To assess the impact of Pol κ deficiency on cisplatin-exposed DRG neurons, Pol κ levels were reduced using siRNA. Pol κ targeting siRNA diminished the cisplatin-induced nuclear Pol κ immunoreactivity in DRG neurons and decreased the extent of cisplatin-induced DNA repair synthesis, as reflected in reduced incorporation of thymidine analog into nuclear DNA. Moreover, Pol κ depletion exacerbated global transcriptional suppression induced by cisplatin in DRG neurons. Collectively, these findings provide the first evidence for critical role of Pol κ in DNA damage response in the nervous system and call attention to implications of polymorphisms that modify Pol κ activity, on maintenance of genomic integrity and neuronal function in exogenously challenged PNS.

Inflammation plays a key role in pancreatitis. Earlier studies from our laboratory showed that experimental pancreatitis activated the pancreatic apelin-APJ axis robustly in mice. Apelin signaling reduced neutrophil invasion and the activation of pancreatic nuclear factor (NF)-κB in mice with experimental pancreatitis. The aim of this study was to assess whether apelin-induced inhibition of pancreatic NF-κB activation was linked mechanistically to apelin's inhibition of pancreatic inflammatory mediator up-regulation in mice with cerulein-induced chronic pancreatitis (CP). Whether apelin's inhibitory effects were associated with the inhibition of NF-κB binding to the promoter region of IL-1β was examined. The effects of apelin exposure on pancreatic IκB degradation/replenishment and membrane levels of phosphorylated protein kinase C were measured. Results demonstrated that apelin inhibited the up-regulation of pancreatic tumor necrosis factor α, macrophage inflammatory protein-1 α/β, and IL-1β expression significantly in mice with CP. Chromatin immunoprecipitation assay findings showed that apelin inhibited NF-κB binding to a putative NF-κB binding site in the IL-1β promoter. Apelin exposure reduced the pancreatic membrane levels of phosphorylated protein kinase C-δ and enhanced the replenishment of pancreatic IκB proteins. Together, these findings indicated that the inhibition of NF-κB activation by apelin was a mechanism behind the reduced pancreatic levels of inflammatory mediators in CP mice exposed to apelin.

Chemotherapy-induced neurotoxicity of peripheral nervous system (PNS) hinders efficacy of cancer treatments. Mechanisms initiating PNS injury by anticancer drugs are incompletely understood delaying development of effective management strategies. To understand events triggered in PNS by cancer drugs, we exposed dorsal root ganglion (DRG) neurons to cisplatin, a drug from platinum-based class of chemotherapeutics frequently implicated in peripheral neuropathies. While cisplatin enters cancer cells and forms cisplatin/DNA crosslinks that block cell proliferation, circulating cisplatin can also reach the PNS and produce crosslinks that impede critical DNA transactions in postmitotic neurons. Cisplatin forms crosslinks with both, nuclear and mitochondrial DNA (mtDNA). Crosslinks are repairable primarily via the nucleotide excision repair (NER) pathway, which is present in nuclei but absent from mitochondrial compartment. Hence, high mitochondrial content and limited shielding by blood nerve barrier make DRG neurons particularly vulnerable to mitochondrial injury by cisplatin. We report that in DRG neurons, cisplatin elevates reactive oxygen species, depletes mtDNA, and impairs mitochondrial respiration, whereas concomitant meclizine supplementation preserves redox balance, attenuates mitochondrial compromise, and augments DNA repair. Meclizine is an antihistamine drug recently implicated in neuroprotection via modulation of energy metabolism. Our data demonstrate that in the mitochondria-rich DRG neurons, meclizine mitigates cisplatin-induced mitochondrial compromise via enhancement of pentose phosphate pathway and repletion of nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione stores. The findings suggest that meclizine-mediated preservation of redox balance sustains mitochondrial respiration and supports execution of cellular processes, including timely removal of cisplatin crosslinks from nuclear DNA, thereby attenuating cisplatin toxicity in DRG neurons. Collectively, the findings reveal potential for pharmacologic modulation of dorsal root ganglion neurons metabolism for protection against toxicity of chemotherapeutic drugs.