Clinical Characterization of Cancer Therapy-induced Adverse Sequelae and Mechanism-based Interventional Strategies

Numerous clinical and preclinical studies have established the debilitating neurocognitive side effects of various chemotherapy regimens for the treatment of cancer, often referred as chemobrain. With substantial increases in the number of cancer survivors, over 16.9 million in the U.S. alone, cognitive function following cancer treatment is considered as one of the most critical criterion for evaluating therapeutic outcome and for determining long-term quality of life. The situation is confounded further by the conspicuous absence of satisfactory treatments for reducing the progressive neurocognitive sequelae associated with non-CNS cancer therapies. This application is in response to a specific RFA (PAR-21-329) to investigate interventions designed to prevent or reduce the adverse neurocognitive sequelae following cancer therapy. Our preclinical studies have shown long-term consequences of chronic chemotherapy (cyclophosphamide, CYP; Adriamycin, ADR monotherapy) including cognitive impairments, loss of neuronal architecture, spine integrity and neuroinflammation. We posit that neuroinflammation is one of the major contributory factors for long-term CNS dysfunction and that human neural stem cell (hNSC)-derived extracellular vesicle (EVs) treatment can ameliorate adverse neurocognitive and inflammatory sequelae associated with chemobrain. Our recent data show that hNSCs or hNSC-derived EV reverse cancer therapy (CYP or irradiation, IRR)-induced cognitive impairments, neuron and spine damage and, neuroinflammation. Intra-venous (retroorbital vein, RO) injections of hNSC-EVs showed long-term neuroprotection in the IRR brain. We have also identified candidate miRNA within the EV cargo, with gene targets relevant to the molecular, structural and behavioral improvements observed in the cancer therapy-exposed animals following EV injection. Importantly, in vivo expression of miR-124-3p reversed IRRinduced cognitive deficits and neuroinflammation. Based on the foregoing, we propose a comprehensive series of studies designed to test the effectiveness hNSC-EV and determine an EV-derived candidate miRNA-based mechanism to ameliorate chemobrain and neuroinflammation in routinely used adjuvant chemotherapy regimens (Carboplatin-Taxol, ADRCYP) to control the growth of ovarian and breast cancer. Our research design will delineate longterm neuroprotective effects of RO injections of hSNC-EV or in vivo expression of miR-124-3p following adjuvant chemotherapy regimens in disease-free or xenograft cancer mouse models. These studies will also elucidate the safety, toxicity and pharmacokinetics of hNSC-EVs therapy in the context of cancer. Thus, this project is based on a foundation of strong published and preliminary data supporting our rationale.

Cancer is the second most frequent cause of death in children under 15 years of age, and primary central nervous system (CNS) tumors are the most frequent cause of cancer-related childhood deaths. Medulloblastoma (MB) is the most frequent malignant childhood brain tumor (incidence of 5.5/million/year). About 40% of cases occur in children <5 years old, which can be sub-divided by biological markers into two groups: low-risk group, biologically defined by either Wingless/Integrated (WNT) or Sonic Hedgehog (SHH) activation TP53-wt, while the high-risk group is defined by non-WNT/non-SHH biology. As WNT-activated MB is extremely rare in early childhood, only young patients (<5 years of age) with low-risk (SHH-activated) MB are eligible and have an excellent prognosis if treated with either of the two randomized arms in this research study. Craniospinal irradiation (CSI) is an integral component in the treatment of MB; however, because of the devastating impact upon the central nervous system (CNS) and neurocognitive outcomes, it must be avoided whenever possible given the significant interference with educational and vocational attainment. Consequently, maintaining or improving neurocognitive and QoL functioning is an essential opportunity for early childhood survivors who can now be cured with treatment that does not include CSI. The Prospective International SIOPE/CONNECT phase-III study to improve neurocognitive outcomes in young children with low-risk medulloblastoma (YCMB-LR) is the first ever randomized study directly comparing two highly effective irradiation-sparing treatment regimens, Head Start 4 and HIT-SKK, which will take place at pediatric oncology centers across Europe and North America and is the first to include neuropsychological and QoL outcome as the primary objective. Aim 1) Compare the overall intelligence and IQ subdomains as measured by the Wechsler Preschool and Primary Scale of Intelligence administered 2.5 years after diagnosis between patients with newly diagnosed, non-metastatic, SHH-activated, TP53-wt MB randomized to the interventional arms A (Head Start 4) or B (HITSKK). Aim 2) Compare the trajectory between the two randomized groups at baseline and again at 2.5 years post diagnosis for: a) overall intelligence and IQ subdomains, b) behavioral development and c) QoL, along with analyses at 2.5 years post diagnosis for: d) fine motor dexterity and processing speed, e) visual-motor integration, f) executive functioning, and g) social-emotional functioning. Aim 3) Several quantitative imaging metrics with regard to brain volumes and white matter injury will serve as ancillary noninvasive biomarkers for comparison of the two interventional arms in Aim 1, and will be statistically correlated with the neurocognitive, QoL and behavioral outcomes in Aim 2. Impact: Our work will define the new “gold standard” of treatment in early childhood low-risk MB that is associated with better neurocognitive outcomes with less severe late-effects and ultimately yield a better QoL in survivorship, while simultaneously improving and harmonizing international diagnostic and therapeutic standards not only for MB, but also for other pediatric CNS tumors.

Hypertension (HTN) is the most common cardiovascular (CV) comorbidity among patients with breast cancer and is an important modifiable risk factor for adverse CV events during and after cancer treatment. Work by our group and others has shown that HTN is an important risk factor for cardiotoxicity caused by curative breast cancer treatments including anthracyclines and human epidermal growth factor receptor 2 (HER2) targeted agents, which occurs in up to 20% of patients receiving these therapies and presents with a reduced left ventricular ejection fraction or heart failure. Furthermore, cardiotoxicity is a leading treatment-limiting toxicity that interferes with curative cancer treatment delivery, worsens cancer outcomes, and leads to persistent impairment of cardiorespiratory fitness in long-term survivors of breast cancer. CV disease is now a leading cause of morbidity and mortality among breast cancer survivors who are living longer due to advances in cancer care, therefore strategies to mitigate CV risk in patients with breast cancer are critically needed. No standard treatment option is currently available to prevent cardiotoxicity during cancer treatment, and no guidelines exist to inform the optimal approach to blood pressure control during cancer treatment. Multiple trials have shown that intensive blood pressure control is associated with CV risk reductions, however exclusion of patients with cancer represents an important limitation of these trials. The association between HTN and cardiotoxicity risk provide a strong rationale for optimizing blood pressure control to improve CV health and reduce cardiotoxicity risk in patients with HTN who are most vulnerable, however no previous trial has assessed the role of intensive blood pressure control on the cardiotoxic effects of breast cancer treatment. The objective of this study is therefore to evaluate intensive systolic blood pressure (SBP) control in women with HTN at risk for cardiotoxicity during BC treatment and the effects of intensive SBP control on biomarkers (imaging, functional, and circulating) of cardiotoxicity. Using a randomized controlled trial design, 130 patients with breast cancer at increased risk for cardiotoxicity (defined by baseline SBP ≥130 mm Hg and treatment with anthracyclines with or without HER2targeted therapy) will be randomly allocated (ratio 1:1) to intensive SBP control (goal SBP <120 mm Hg) versus standard SBP control (goal SBP <140 mm Hg) prior to initiating breast cancer treatment. Aim 1: Evaluate the efficacy of an intensive SBP control intervention during active BC treatment in patients at risk for cardiotoxicity. Aim 2: Evaluate the effects of intensive SBP control on imaging and functional biomarkers of cardiotoxicity. Aim 3: Assess the effect of intensive SBP control on circulating biomarkers of cardiotoxicity. The results from this investigation will: 1) establish critical data to inform clinical implementation of intensive SBP control for patients with breast cancer at risk for cardiotoxicity, 2) provide functional and mechanistic insights into the effects of intensive SBP control on mitigation of cardiotoxicity risk, and 3) guide future cardio-oncology practice recommendations on the role of HTN management to improve CV health in patients with cancer.

Chemotherapy-induced peripheral neuropathy (CIPN) is a serious side effect that causes morbidity and limits the dose of chemotherapy allowed to treat cancers. In most classes of neurotoxic chemotherapeutics, CIPN manifests as damage to dorsal root ganglia sensory neurons. The neuronal damage is thought to be due to an unelucidated combination of dysfunctional microtubules, axonal transport and mitochondria that ultimately leads to programmed axonal degeneration via a nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) and Sterile Alpha and TIR Motif Containing 1 (SARM1) pathway. The link between these processes, however, remains entirely unknown. Using a human induced pluripotent stem cell (iPSC)-derived dorsal root ganglia sensory neuronal culture system (iSN), we have identified microtubule-associated protein 2 (MAP2) as a key determinant of CIPN. When exposed to clinically-relevant doses of bortezomib, paclitaxel or vincristine, MAP2 levels drop and there is subcellular MAP2 mislocalization in iSN that occurs prior to axonal degeneration. Critically, overexpression of MAP2 is protective for bortezomib-induced neurotoxicity in iSN. In this grant application, we propose to further dissect the role of MAP2 in CIPN by utilizing an innovative CRISPR-ErCas12a system to develop genetically-engineered iPSC lines that fluorescently-tag either MAP2 or NMNAT2. These novel iPSC lines will allow for detailed analyses of the time course of altered subcellular localization and axonal transport, and how these processes link to SARM1 activation and axonal degeneration. Furthermore, we will investigate the overlapping mechanisms in CIPN due to bortezomib, paclitaxel, and vincristine using our previously successful proteomics analysis approach, which now will employ a state-of-the-art quantitative proteomic and phosphoproteomic technology. The proposed Specific Aims build on existing understanding of the pathomechanisms of CIPN while innovating into unexplored novel areas that are potential targets for preventative therapeutic development.

Cancer-related cognitive impairment (CRCI), often referred as “chemobrain”, is prevalent up to 75% of all breast cancer survivors. These impairments cause significant distress and reduce the quality for life for survivors. Despite growing realization of the long-term clinical problem of CRCI in millions of cancer survivors, there is a conspicuous absence of clinical recourse available. Therefore, regenerative strategies to restore cognition and normal brain function in the cancer patients and survivors are clearly needed. Our past clinical and pre-clinical studies have established that doxorubicin, which is commonly used in breast cancer, can lead to a significant decline in the blood (human) and brain (rodent) levels of brain derived neurotrophic factor (BDNF). BDNF is abundantly expressed in the prefrontal cortex and hippocampus and plays important roles in neuronal repair and survival, dendritic and axonal growth, long-term potentiation, and neural stem cell maintenance. In our human studies, pathological reductions of BDNF were linked to higher risk of cognitive toxicity. Similarly, we have shown that chronic chemotherapy significantly impaired performance on the hippocampus and cortex-dependent cognitive tasks in the rodents. These deficits were linked with reduced neurogenesis, elevated neuroinflammation, and significant damage to the newly born and mature neuronal architecture, dendrites, spines, and synaptic integrity. To mitigate these deficits, our preliminary rodent studies involving mice receiving doxorubicin with riluzole, an orally active glutamate-modulating medication, has prevented the reduction of hippocampus BDNF levels. Thus, we hypothesize that: i) chemotherapy-induced reduction of BDNF leads to the long-term neurodegenerative consequences culminating into cognitive impairments and, ii) augmentation of BDNF in vivo will restore cognitive function in brains exposed to chemotherapy and will provide neuroprotection against CRCI. We will test our hypothesis with three specific aims. In Aim 1, we will systematically examine brain and plasma BDNF levels to link its trajectory with CRCI and neurobiological underpinnings in a mouse model of breast cancer chemotherapy. In Aim 2, we will determine the neuroprotective impact of enhancing BDNF in vivo to reverse CRCI. In Aim 3, we will evaluate the neuroprotective effect of BDNF-enhancing riluzole to ameliorate CRCI. This study will link the neurobiological underpinnings of chemotherapy and neuroprotective effects of BDNF against CRCI. If demonstrated to be successful, our translationally feasible pharmacological approach will provide basis for future studies to repurpose riluzole as a therapeutic option for mitigating CRCI.

Breast cancer is the second most common cancer among women in the U.S., with most cases diagnosed among postmenopausal women at an early and treatable stage. The majority of tumors are hormone-receptor positive and patients receive adjuvant endocrine treatment with aromatase inhibitors (AI) to prolong diseasefree survival and time-to-recurrence. Unfortunately, AI-associated musculoskeletal symptoms (AIMSS) such as joint pain and muscle stiffness/achiness is a common side-effect of AIs, which causes approximately onefourth of patients discontinue their therapy. The precise mechanisms of AIMSS are unknown and no therapies are approved for prevention or treatment. There is clearly an urgent need to identify and validate novel targets to facilitate development of new treatments that are effective and safe. This proposal focuses on a promising target: the sphingosine-1-phosphate type-1 receptor (S1PR1). Our preliminary data suggest for the first time that S1P contributes to AIMSS-related effects produced by repeated oral administration of letrozole, a widely used AI, in female mice. Letrozole treatment increased levels of S1P in the lumbar spinal cord in female ovariectomized mice. Furthermore, letrozole-induced AIMSS-related symptoms were completely absent in conditional null mice lacking S1PR1 in CNS cell lineages compared to control mice. The effect of FTY720, which is an FDA-approved S1PR1/3/4/5 agonist prodrug, was then assessed as a potential treatment in our model. Oral FTY720 administration reversed letrozole-induced pain-like behaviors and functional impairment in a dose- and time-dependent manner. Treatment with FTY720 also rapidly desensitized S1PR1 signaling in the CNS, suggesting a functional antagonist mechanism of action. Collectively, our preliminary results suggest that S1PR1 represents a promising novel target for the treatment of AIMSS. This project will test the central hypothesis that S1PR1 activation, mainly in astrocytes, contributes to letrozole-induced AIMSS-related symptoms and that competitive or functional antagonism of S1PR1 alleviates these effects. Aim 1 will determine whether competitive antagonism of S1PR1 will alleviate and prevent letrozole-induced AIMSS-related symptoms. Aim 2 will determine whether the S1PR1-selectively agonist, ponesimod, will functionally antagonize SPR1 by desensitization or downregulation of S1PR1 in the CNS to alleviate and prevent AIMSS symptoms. We will also ensure that these S1PR1 ligands do not interfere with the anti-aromatase activity of letrozole in in vitro and in vivo breast cancer models. Aim 3 will determine the role of S1PR1 in specific cell types (astrocytes, neurons, and microglia/macrophages) in letrozole-induced AIMSS. Overall, this project aims to elucidate the target receptor type, cell type(s) and pharmacological mechanism responsible for S1PR1 modulator-induced reversal of AIMSS, thereby providing a rationale for development of S1PR1-based medications to treat this side effect of cancer adjuvant treatment.