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Table S1: Oligonucleotide primer sequences. Table S2: Data obtained from the Cox model. Figure S1: Detailed structure of the human TRIM29 gene locus. Figure S2: Additional TRIM29 gene methylation and transcript abundance analysis. Figure S3: Analysis of gene expression in breast cancer cells with TRIM29 knockdown. Figure S4: qRT-PCR analysis of TWIST1 and N-cadherin mRNA abundance in TRIM29 knockdown and control SK-BR-3 cells. Figure S5: Results of multivariate survival analysis examining TRIM29 expression vs patient age (A), lymph node status (B), size (C), Grade (D), and ER status (E).

Introduction: Netherton syndrome (NS) is a rare multisystem disorder with both cutaneous and extra-cutaneous manifestations. The syndrome includes the presence of congenital ichthyosis, exhibited by hyperkeratosis and abnormal skin scaling over the entire body. In rare instances, NS has been associated with the development of squamous cell carcinoma (SCC). Case Report: A 54-year-old male with a history of NS and recurrent squamous cell carcinoma presented with an enlarging left inguinal mass. Biopsy of the mass was positive for a new invasive squamous cell carcinoma with regional lymph node involvement and invasion of the left femoral vein. The patient underwent wide local resection, superficial and deep regional lymph node dissection, and resection of the left femoral vein. His hospital course was further complicated by multiple infections and progression of his cancer. He eventually transitioned to hospice care before receiving any additional treatment for his malignancy. Conclusion: This case highlights a patient with NS and a history of recurrent inguinal squamous cell carcinoma. Further investigation is needed to better highlight the complexity of factors associated with the development of squamous cell carcinoma in patients with NS and other forms of ichthyoses.

Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal recessive lysosomal disease caused by defective production of the enzyme α-N-acetylglucosaminidase. It is characterized by severe and complex central nervous system degeneration. Effective therapies will likely target early onset disease and overcome the blood–brain barrier. Modifications of adeno-associated viral (AAV) vector capsids that enhance transduction efficiency have been described in the retina. Herein, we describe for the first time, a transduction assessment of two intracranially administered adeno-associated virus serotype 8 variants, in which specific surface-exposed tyrosine (Y) and threonine (T) residues were substituted with phenylalanine (F) and valine (V) residues, respectively. A double-mutant (Y444 + 733F) and a triple-mutant (Y444 + 733F + T494V) AAV8 were evaluated for their efficacy for the potential treatment of MPS IIIB in a neonatal setting. We evaluated biodistribution and transduction profiles of both variants compared to the unmodified parental AAV8, and assessed whether the method of vector administration would modulate their utility. Vectors were administered through four intracranial routes: six sites (IC6), thalamic (T), intracerebroventricular, and ventral tegmental area into neonatal mice. Overall, we conclude that the IC6 method resulted in the widest biodistribution within the brain. Noteworthy, we demonstrate that GFP intensity was significantly more robust with AAV8 (double Y–F + T–V) compared to AAV8 (double Y–F). This provides proof of concept for the enhanced utility of IC6 administration of the capsid modified AAV8 (double Y–F + T–V) as a valid therapeutic approach for the treatment of MPS IIIB, with further implications for other monogenic diseases.

Background During their work on the cerebrospinal fluid (CSF) circulatory system of human nerves and brain, the authors applied imaging and tissue techniques that complemented basic anatomical dissection. Objectives The authors sought to show how integrating fluorescent imaging and basic immunohistochemistry (IHC) with facial anatomy can address current problems in aesthetic surgery. Methods The authors developed an algorithm and a set of principles from their work on the CSF circulatory system and applied these to 3 problems in aesthetic surgery: the functional anatomy of the vermilion-cutaneous junction; chemosis; and the functional anatomy of periosteal fixation. Results Integrating fluorescent imaging and IHC with anatomical dissection characterizes structural and functional anatomy. Fluorescent imaging helps to identify and locate easily missed structures. IHC defines cell type and function. The vermilion-cutaneous junction is defined by a major lymphatic vessel. Lymphatic flow from the medial limbus to the lateral canthus suggests the etiology of chemosis. Periosteal sites of fixation prevent shear where dural CSF vessels drain directly to subcutaneous lymphatics. Conclusions Integrating anatomical dissection with fluorescent imaging and basic IHC characterizes structural and functional anatomy and helps to better understand many problems encountered in aesthetic surgery.

Since it is extremely difficult to establish an animal model for human chromosomal abnormalities, induced pluripotent stem cells (iPSCs) provide a powerful alternative to study underlying mechanisms of these disorders and identify potential therapeutic interventions. In this study we established iPSCs from a young girl with a hemizygous deletion of Xq27.3-q28 who exhibited global developmental delay and intellectual disability from early in infancy. The deletion site on the X chromosome includes Fragile X Mental Retardation 1 (FMR1), the gene responsible for fragile X syndrome, which likely contributes to the patient's neurodevelopmental abnormalities. The FMR1 gene was expressed in approximately half of the iPSC clones we generated while it was absent in the other half due to the random inactivation of normal and abnormal X chromosomes. The normal or absent expression pattern of the FMR1 gene was not altered when the iPSCs were differentiated into neural progenitor cells (NPCs). Moreover, chromosome reactivating reagents such as 5-aza-2-deoxycytidine, trichostatin A, and UNC0638, were tested in an attempt to reactivate the suppressed FMR1 gene in affected iPSC-NPCs. The affected and control isogenic iPSCs developed in this study are ideal models with which to identify downstream consequences caused by the Xq27.3-q28 deletion and also to provide tools for high-throughput screening to identify compounds potentially improving the well-being of this patient population.

Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal recessive lysosomal storage disease caused by mutations in the gene that encodes the protein N-acetyl-glucosaminidase (NAGLU). Defective NAGLU activity results in aberrant retention of heparan sulfate within lysosomes leading to progressive central nervous system (CNS) degeneration. Intravenous treatment options are limited by the need to overcome the blood–brain barrier and gain successful entry into the CNS. Additionally, we have demonstrated that AAV8 provides a broader transduction area in the MPS IIIB mouse brain compared with AAV5, 9 or rh10. A triple-capsid mutant (tcm) modification of AAV8 further enhanced GFP reporter expression and distribution. Using the MPS IIIB mouse model, we performed a study using either intracranial six site or intracisterna magna injection of AAVtcm8-codon-optimized (co)-NAGLU using untreated MPS IIIB mice as controls to assess disease correction. Disease correction was evaluated based on enzyme activity, heparan sulfate storage levels, CNS lysosomal signal intensity, coordination, activity level, hearing and survival. Both histologic and enzymatic assessments show that each injection method results in supranormal levels of NAGLU expression in the brain. In this study, we have shown correction of lifespan and auditory deficits, increased CNS NAGLU activity and reduced lysosomal storage levels of heparan sulfate following AAVtcm8-coNAGLU administration and partial correction of NAGLU activity in several peripheral organs in the murine model of MPS IIIB.

Breast cancer mortality remains unacceptably high, indicating a need for safer and more effective therapeutic agents. Disulfide bond Disrupting Agents (DDAs) were previously identified as a novel class of anticancer compounds that selectively kill cancers that overexpress the Epidermal Growth Factor Receptor (EGFR) or its family member HER2. DDAs kill EGFR+ and HER2+ cancer cells via the parallel downregulation of EGFR, HER2, and HER3 and activation/oligomerization of Death Receptors 4 and 5 (DR4/5). However, the mechanisms by which DDAs mediate these effects are unknown. Affinity purification analyses employing biotinylated-DDAs reveal that the Protein Disulfide Isomerase (PDI) family members AGR2, PDIA1, and ERp44 are DDA target proteins. Further analyses demonstrate that shRNA-mediated knockdown of AGR2 and ERp44, or expression of ERp44 mutants, enhance basal and DDA-induced DR5 oligomerization. DDA treatment of breast cancer cells disrupts PDIA1 and ERp44 mixed disulfide bonds with their client proteins. Together, the results herein reveal DDAs as the first small molecule, active site inhibitors of AGR2 and ERp44, and demonstrate roles for AGR2 and ERp44 in regulating the activity, stability, and localization of DR4 and DR5, and activation of Caspase 8.

TPS280 Background: Patients with advanced colorectal cancer after progressing through first line therapy, have several FDA-approved systemic therapies that are associated with clinical benefit for a substantive minority of patients. Current clinical practice is to trial these various treatments in a step-wise fashion using CT scans every 3 months to evaluate effectiveness. This process requires 3-4 months between therapeutic interventions from which the patient may ultimately derive no clinical benefit, may have a performance status decline; limiting the number of possible interventions and increases risk for physical and financial toxicity. An alternative to the traditional CT-scan guided approach for disease assessment is a circulating tumor DNA (ctDNA) intervention. The Signatera ctDNA assay which utilizes 16 truncal mutations derived from a patient’s tumor, can be assessed every 2 weeks for a rapid determination of the effectiveness of a systemic therapy. This may allow patients to be exposed to many treatments during a short time, limiting toxicity, allowing for a quicker determination of clinical benefit and personalization of treatment. The aim of this study will be to compare the traditional scan-driven approach vs an intervention guided by ctDNA assessments, both arms using a pre-specified order of chemotherapy treatments. Methods: This is a phase 2 randomized study of patients with refractory metastatic adenocarcinoma of the colon or rectum. Participants are eligible after progression or intolerance to first line chemotherapy or recurrence within 6 months of adjuvant oxaliplatin based chemotherapy. They must have RECIST measurable metastatic disease that is not eligible for definitive management. Tissue from the primary and/or metastatic deposit is required for Signatera NGS analysis and subjects must have measurable ctDNA at sampling. Participants must be ≥18 years old without major organ dysfunction and have an ECOG performance status of 0 to 2. Subjects with Microsatellite High, deficient in DNA mismatch repair genes, or BRAF V600E mutations are excluded. Subjects will be randomized 1:1 to Arm A (ctDNA guided intervention) or Arm B (scan-guided control group). Patients in both arms will undergo systemic treatments in a standardized pre-specified order. Arm A will have ctDNA assessments every 2 weeks until an intervention shows a significant decrease, then every 4 weeks until Progressive Disease (PD) by scan or significant ctDNA increase. CT imaging will be performed every 12 weeks. Those in Arm B, will have CT imaging every 12 weeks and blood collected for post-hoc analysis every 4 weeks until PD by scan. The primary endpoint is overall survival. Secondary endpoints include progression free survival and overall response. Exploratory analysis with be performed of the microbiome. Enrollment continues to a maximum of 78 patients. Clinical trial information: NCT04786600 .

Tranexamic Acid (TA) is a clinically used antifibrinolytic that acts as a lysine mimetic to block binding of Plasminogen with Plasminogen activators, preventing conversion of Plasminogen to its proteolytically activated form, Plasmin. Previous studies suggested that TA may exhibit anticancer activity by blockade of extracellular Plasmin formation. Plasmin-mediated cleavage of the CDCP1 protein may increase its oncogenic functions through several downstream pathways. Results presented herein demonstrate that TA blocks Plasmin-mediated excision of the extracellular domain of the oncoprotein CDCP1. In vitro studies indicate that TA reduces the viability of a broad array of human and murine cancer cell lines, and breast tumor growth studies demonstrate that TA reduces cancer growth in vivo. Based on the ability of TA to mimic lysine and arginine, we hypothesized that TA may perturb multiple processes that involve Lys/Arg-rich protein sequences, and that TA may alter intracellular signaling pathways in addition to blocking extracellular Plasmin production. Indeed, TA-mediated suppression of tumor cell viability is associated with multiple biochemical actions, including inhibition of protein synthesis, reduced activating phosphorylation of STAT3 and S6K1, decreased expression of the MYC oncoprotein, and suppression of Lys acetylation. These findings suggest that TA or TA analogs may serve as lead compounds and inspire the production of new classes of anticancer agents that function by mimicking Lys and Arg.

Tranexamic Acid (TA) is a clinically used antifibrinolytic agent that acts as a Lys mimetic to block binding of Plasminogen with Plasminogen activators, preventing conversion of Plasminogen to its proteolytically activated form, Plasmin. Previous studies suggested that TA may exhibit anticancer activity by blockade of extracellular Plasmin formation. Plasmin-mediated cleavage of the CDCP1 protein may increase its oncogenic functions through several downstream pathways. Results presented herein demonstrate that TA blocks Plasmin-mediated excision of the extracellular domain of the oncoprotein CDCP1. In vitro studies indicate that TA reduces the viability of a broad array of human and murine cancer cell lines, and breast tumor growth studies demonstrate that TA reduces cancer growth in vivo. Based on the ability of TA to mimic Lys and Arg, we hypothesized that TA may perturb multiple processes that involve Lys/Arg-rich protein sequences, and that TA may alter intracellular signaling pathways in addition to blocking extracellular Plasmin production. Indeed, TA-mediated suppression of tumor cell viability is associated with multiple biochemical actions, including inhibition of protein synthesis, reduced activating phosphorylation of STAT3 and S6K1, decreased expression of the MYC oncoprotein, and suppression of Lys acetylation. Further, TA inhibited uptake of Lys and Arg by cancer cells. These findings suggest that TA or TA analogs may serve as lead compounds and inspire the production of new classes of anticancer agents that function by mimicking Lys and Arg.

Disulfide bond-disrupting agents (DDAs) are a new chemical class of agents recently shown to have activity against breast tumors in animal models. Blockade of tumor growth is associated with downregulation of EGFR, HER2, and HER3 and reduced Akt phosphorylation, as well as the induction of endoplasmic reticulum stress. However, it is not known how DDAs trigger cancer cell death without affecting nontransformed cells. As demonstrated here, DDAs are the first compounds identified that upregulate the TRAIL receptor DR5 through transcriptional and post-transcriptional mechanisms to activate the extrinsic cell death pathway. At the protein level, DDAs alter DR5 disulfide bonding to increase steady-state DR5 levels and oligomerization, leading to downstream caspase 8 and 3 activation. DDAs and TRAIL synergize to kill cancer cells and are cytotoxic to HER2+ cancer cells with acquired resistance to the EGFR/HER2 tyrosine kinase inhibitor Lapatinib. Investigation of the mechanisms responsible for DDA selectivity for cancer cells reveals that DDA-induced upregulation of DR5 is enhanced in the context of EGFR overexpression. DDA-induced cytotoxicity is strongly amplified by MYC overexpression. This is consistent with the known potentiation of TRAIL-mediated cell death by MYC. Together, the results demonstrate selective DDA lethality against oncogene-transformed cells, DDA-mediated DR5 upregulation, and protein stabilization, and that DDAs have activity against drug-resistant cancer cells. Our results indicate that DDAs are unique in causing DR5 accumulation and oligomerization and inducing downstream caspase activation and cancer cell death through mechanisms involving altered DR5 disulfide bonding. DDAs thus represent a new therapeutic approach to cancer therapy.

BACKGROUND: Breast cancer-related lymphedema (BCRL) is a source of postoperative morbidity for breast cancer survivors. Lymphatic microsurgical preventive healing approach (LYMPHA) is a technique used to prevent BCRL at the time of axillary lymph node dissection (ALND). We report the 5-year experience of a breast surgeon trained in LYMPHA and investigate the outcomes of patients who underwent LYMPHA following ALND for the treatment of cT1-4N1-3M0 breast cancer. METHODS: A retrospective review of patients with T1-4N1-3M0 breast cancer was performed in patients who underwent ALND with and without LYMPHA. Diagnosis of BCRL was made by certified lymphedema therapists. Descriptive statistics and lymphedema surveillance data were analyzed using results of Fisher’s exact or Wilcoxon rank sum tests. Logistic regression and propensity matching were performed to assess the reduction of BCRL occurrence following LYMPHA. RESULTS: In a 5-year period, 132 patients met inclusion criteria with 76 patients undergoing LYMPHA at the time of ALND and 56 patients undergoing ALND alone. Patients who underwent LYMPHA at the time of ALND were significantly less likely to develop BCRL than those who underwent ALND alone (p=0.045). Risk factors associated with BCRL development were increased patient age (p=0.007), BMI (p=0.003), and, in patients undergoing LYMPHA, the number of positive nodes (p=0.026). CONCLUSIONS: LYMPHA may be successfully employed by breast surgeons trained in lymphatic-venous anastomosis at the time of ALND. While research efforts should continue to focus on the prevention and surveillance of BCRL, LYMPHA remains an option to reduce BCRL and improve patient quality of life.

Despite the reduced caloric content of artificially sweetened beverages (ASBs) relative to those sweetened with sucrose, consumption of ASBs fail to consistently decrease the risk of obesity and associated diseases. This failure may be due to the inability of ASBs to effectively reduce appetite and hence overall caloric intake. A variety of non-nutritive sweeteners (NNS), however, remain to be screened for effectiveness in promoting satiety and reducing calorie consumption. Erythritol is well-tolerated, low-calorie sugar alcohol widely used as a sugar substitute. It is unique among NNS due to its low sweetness index relative to glucose, meaning that it is typically served at much higher concentrations than other common NNS. Animal and human studies have noted correlations between osmolarity, satiety, and levels of satiety hormones, independent of the effects of sweetness or nutritive value. We hypothesized that consumption of a beverage sweetened with erythritol to the sweetness and osmolarity of a common soft drink will improve self-reported satiety and more strongly affect the magnitude of changes in the hormone ghrelin than would an iso sweet beverage sweetened only with aspartame, a sweetener with a high sweetness index relative to glucose. Using a randomized double-blind crossover trial, we found that serum ghrelin was significantly decreased after consumption of an erythritol-sweetened beverage compared to aspartame. Likewise, consumption of the erythritol-sweetened beverage increased various measures of satiety in volunteers. Knowledge gained from this project demonstrates that high-osmolarity NNS may be useful in formulating ASBs that are satiating and low in calories.

While HER2 and EGFR are overexpressed in breast cancers and multiple other types of tumors, the use of EGFR and/or HER2 inhibitors have failed to cure many cancer patients, largely because cancers acquire resistance to HER2/EGFR-specific drugs. Cancers that overexpress the HER-family proteins EGFR, HER2, and HER3 are uniquely sensitive to agents that disrupt HER2 and EGFR protein folding. We previously showed that disruption of disulfide bond formation by Disulfide Disrupting Agents (DDAs) kills HER2/EGFR overexpressing cells through multiple mechanisms. Herein, we show that interference with proline isomerization in HER2/EGFR overexpressing cells also induces cancer cell death. The peptidyl-prolyl isomerase inhibitor Cyclosporine A (CsA) selectively kills EGFR+ or HER2+ breast cancer cells in vitro by activating caspase-dependent apoptotic pathways. Further, CsA synergizes with the DDA tcyDTDO to kill HER2/EGFR overexpressing cells in vitro and the two agents cooperate to kill HER2+ tumors in vivo. There is a critical need for novel strategies to target HER2+ and EGFR+ cancers that are resistant to currently available mechanism-based agents. Drugs that target HER2/EGFR protein folding, including DDAs and CsA, have the potential to kill cancers that overexpress EGFR or HER2 through the induction of proteostatic synthetic lethality.

BACKGROUND Obesity has been associated with attenuated vaccine responses and an increased risk of contracting pneumococcal pneumonia, but no study to our knowledge has assessed the impact of obesity and genetics on 23-valent pneumococcal vaccine (PPSV23) efficacy. We assessed the relationship of obesity (primary analysis) and stimulator of interferon genes (STING1) genotype (secondary analysis) on PPSV23 efficacy. METHODS Nonobese (BMI 22–25 kg/m2) and obese participants (BMI ≥30 kg/m2) were given a single dose of PPSV23. Blood was drawn immediately prior to and 4–6 weeks after vaccination. Serum samples were used to assess PPSV23-specific antibodies. STING1 genotypes were identified using PCR on DNA extracted from peripheral blood samples. RESULTS Forty-six participants were categorized as nonobese (n = 23; 56.5% women; mean BMI 23.3 kg/m2) or obese (n = 23; 65.2% women; mean BMI 36.3 kg/m2). Obese participants had an elevated fold change in vaccine-specific responses compared with nonobese participants (P < 0.0001). The WT STING1 group (R232/R232) had a significantly higher PPSV23 response than individuals with a single copy of HAQ-STING1 regardless of BMI (P = 0.0025). When WT was assessed alone, obese participants had a higher fold serotype-specific response compared with nonobese participants (P < 0.0001), but no difference was observed between obese and nonobese individuals with 1 HAQ allele (P = 0.693). CONCLUSIONS These observations demonstrate a positive association between obesity and PPSV23 efficacy specifically in participants with the WT STING1 genotype. TRIAL REGISTRATION ClinicalTrials.gov NCT02471014. FUNDING This research was supported by the NIH and the University of Florida MD-PhD Training Program., Obesity and a WT STING1 genotype are positively associated with efficacy of the 23-valent pneumococcal vaccine in a small cohort of subjects.

This article presents current best knowledge to assess the projected outcomes benefit of adding multi-modality surveillance imaging to standard follow-up care for breast cancer patients at high risk (>30%) for developing future metastases. This analysis is motivated by recent preliminary clinical studies that have suggested that augmenting systemic treatment of early-stage metastases with targeted surgery and/or radiosurgery achieves significant overall survival and disease-free survival benefit. Our primary aims are to: (a) describe the clinical motivation and scan parameters needed to identify the early onset of metastatic progression in breast cancer patients for effective surgical or radiosurgical treatment; (b) estimate the anticipated survival benefit for high-risk patients under this recommended protocol; and (c) estimate the radiation risks associated with the repeated body imaging of this protocol.

TPS223 Background: Patients with advanced colorectal cancer after progressing through first line therapy, have several FDA-approved systemic therapies that are associated with clinical benefit for a substantive minority of patients. Current clinical practice is to trial these various treatments in a step-wise fashion using CT scans every 3 months to evaluate effectiveness. This process requires 3-4 months between therapeutic interventions from which the patient may ultimately derive no clinical benefit, may have a performance status decline; limiting the number of possible interventions and increases risk for physical and financial toxicity. An alternative to the traditional CT-scan guided approach for disease assessment is a circulating tumor DNA (ctDNA) intervention. The Signatera ctDNA assay which utilizes 16 truncal mutations derived from a patient’s tumor, can be assessed every 2 weeks for a rapid determination of the effectiveness of a systemic therapy. This may allow patients to be exposed to many treatments during a short time, limiting toxicity, allowing for a quicker determination of clinical benefit and personalization of treatment. The aim of this study will be to compare the traditional scan-driven approach vs an intervention guided by ctDNA assessments, both arms using a pre-specified order of chemotherapy treatments. Methods: This is a phase 2 randomized study of patients with refractory metastatic adenocarcinoma of the colon or rectum. Participants are eligible after progression or intolerance to first line chemotherapy or recurrence within 6 months of adjuvant oxaliplatin based chemotherapy. They must have RECIST measurable metastatic disease that is not eligible for definitive management. Tissue from the primary and/or metastatic deposit is required for Signatera NGS analysis and subjects must have measurable ctDNA at sampling. Participants must be ≥18 years old without major organ dysfunction and have an ECOG performance status of 0 to 2. Subjects with Microsatellite High, deficient in DNA mismatch repair genes, or BRAF V600E mutations are excluded. Subjects will be randomized 1:1 to Arm A (ctDNA guided intervention) or Arm B (scan-guided control group). Patients in both arms will undergo systemic treatments in a standardized pre-specified order. Arm A will have ctDNA assessments every 2 weeks until an intervention shows a significant decrease, then every 4 weeks until Progressive Disease (PD) by scan or significant ctDNA increase. CT imaging will be performed every 12 weeks. Those in Arm B, will have CT imaging every 12 weeks and blood collected for post-hoc analysis every 4 weeks until PD by scan. The primary endpoint is overall survival. Secondary endpoints include progression free survival and overall response. Exploratory analysis with be performed of the microbiome. Enrollment continues to a maximum of 78 patients. Clinical trial information: NCT04786600.

Key Points Question What were the utilization trends associated with granulocyte colony-stimulating factors for primary prophylaxis of febrile neutropenia (FN) among patients with cancer receiving myelosuppressive chemotherapy from 2014 to 2019? Findings This cross-sectional study of 119 129 chemotherapy courses found that among commercially and federally insured populations, use of granulocyte colony-stimulating factors increased among patients receiving high–FN risk regimens, from 75% to 83% for the commercially insured population and from 75% to 86% for the Medicare population. Meaning This study found that there were increases in the use of granulocyte colony-stimulating factors among patients with high FN risk, but 14% to 17% of patients with high FN risk still did not receive preventive treatment., This cross-sectional study assesses granulocyte colony-stimulating factor utilization trends among US adults with cancer overall and stratified by regimen febrile neutropenia risk level., Importance The introduction of biosimilars and novel delivery devices between 2014 and 2019 may have changed the utilization of granulocyte colony-stimulating factors (G-CSF). Objective To assess utilization trends of G-CSFs for primary prophylaxis of febrile neutropenia (FN) among patients with cancer receiving myelosuppressive chemotherapy with commercial or Medicare insurance. Design, Setting, and Participants This cross-sectional study assessed G-CSF utilization trends overall and stratified by regimen febrile neutropenia risk level. Associations between patient characteristics and G-CSF use were evaluated. Patients with cancer, including breast, lung, colorectal, esophageal and gastric, pancreatic, prostate, ovarian, and non-Hodgkin lymphomas, initiating myelosuppressive chemotherapy courses were included from the 2014 to 2019 commercial insurance and 2014 to 2018 Medicare fee-for-service claims databases. Data were analyzed from March to June 2021. Exposures Year of chemotherapy initiation. Main Outcomes and Measures The main outcomes were use and trends of G-CSFs for primary prophylaxis, from completion to 3 days after in the first chemotherapy cycle. Results In total, 86 731 chemotherapy courses (mean [SD] age, 57.7 [11.5] years; 57 838 [66.7%] women and 28 893 [33.3%] men) were identified from 82 410 patients in the commercial insurance database and 32 398 chemotherapy courses (mean [SD] age, 71.8 [8.3] years; 18 468 [57.0%] women and 13 930 [43.0%] men) were identified from 30 279 patients in the Medicare database. Among the commercially insured population, 39 639 patients (45.7%) received G-CSFs, and 12 562 patients (38.8%) received G-CSFs among Medicare insured patients. Overall G-CSF use increased significantly throughout the study period in both populations, from 45.1% (95% CI, 44.4%-45.7%) of patients in 2014 to 47.5% (95% CI, 46.5%-48.5%) of patients in 2019 (P = .001) in the commercially insured population and from 36.0% (95% CI, 34.2%-38.0%) of patients in 2014 to 39.1% (95% CI, 38.1%-40.1%) of patients in 2018 (P

Background Low satisfaction rates and severe complications are two major limitations for temporal hollowing augmentation using autologous fat grafting. Despite fat compartments in temporal region have been reported, its clinical applied anatomy for fat grafting have not been the subject of studies that show its benefits objectively and statistically. Objectives To investigate temporal fat compartments and relative neurovascular structures in cadavers, developing a safe and effective fat grafting technique for temporal hollowing augmentation. Methods The study was conducted on 8 cadavers (16 temples). The tissue layers, fat compartments, ligaments, and neurovascular structures in the temporal region were analysed. The variables were the number and location of sentinel veins, perforator vessels of the middle temporal vein. Measurements were taken with a digital calliper. Results Two separate fat compartments, the lateral temporal-cheek fat compartment and lateral orbital fat compartment, were found in the subcutaneous layer, and two separate septum compartments, the upper and lower temporal compartment, were found in the loose areolar tissue layer. One sentinel vein and 1 to 6 perforator vessels were found to travel through the subcutaneous tissue layer, traverse the overlapping tissue layers in the lower temporal septum region, and finally join in the middle temporal vein. Conclusions The four fat compartments in the temporal region are ideal receipt sites for fat grafting. The medial border of the junction of the hairline and temporal line is a safe and effective cannula entry site for temporal fat grafting. The anterior half of the lower temporal compartment is a “zone of caution” for temporal fat grafting.

Background The experiences of African American adult patients before, during, and after acute care utilization are not well characterized for individuals with sickle cell disease (SCD) or cancer. Objective To describe the experiences of African Americans with SCD or cancer before, during, and after hospitalization for pain control. Methods We conducted a qualitative study among African American participants with SCD (n = 15; 11 male; mean age, 32.7 ± 10.9 years; mean pain intensity, 7.8 ± 2.6) or cancer (n = 15; 7 male; mean age, 53.7 ± 15.2 years; mean pain intensity, 4.9 ± 3.7). Participants completed demographic questions and pain intensity using PAINReportIt and responded to a 7-item open-ended interview, which was recorded and transcribed verbatim. We used content analysis to identify themes in the participants' responses. Results Themes identified included reason for admission, hospital experiences, and discharge expectations. Pain was the primary reason for admission for participants with SCD (n = 15) and for most participants with cancer (n = 10). Participants of both groups indicated that they experienced delayed treatment and a lack of communication. Participants with SCD also reported accusations of drug-seeking behavior, perceived mistreatment, and feeling of not being heard or believed. Participants from both groups verbalized concerns about well-being after discharge and hopeful expectations. Conclusions Race-concordant participants with SCD but not with cancer communicated perceived bias from healthcare providers. Implications for practice Practice change interventions are needed to improve patient-provider interactions, reduce implicit bias, and increase mutual trust, as well as facilitate more effective pain control, especially for those who with SCD.

Disulfide bond Disrupting Agents (DDAs) are a new chemical class of agents recently shown to have activity against breast tumors in animal models. However, it is unknown how DDAs trigger cancer cell death without affecting nontransformed cells. As demonstrated here, DDAs are the first compounds identified that upregulate the TRAIL receptor DR5 through both transcriptional and posttranscriptional mechanisms. At the protein level, DDAs alter DR5 disulfide bonding to increase steady-state DR5 levels and oligomerization, leading to downstream Caspase 8 and 3 activation. DDAs and TRAIL synergize to kill cancer cells and are cytotoxic to HER2+ cancer cells with acquired resistance to the EGFR/HER2 tyrosine kinase inhibitor. Investigation of the mechanisms responsible for DDA selectivity for cancer cells reveals that DDA-induced upregulation of DR5 is enhanced in the context of EGFR overexpression, and DDA-induced cytotoxicity is strongly amplified by MYC overexpression. Together, the results demonstrate selective DDA lethality against oncogene-transformed cells, DDA-mediated DR5 upregulation and protein stabilization, and DDAs against drug-resistant and metastatic cancer cells. DDAs thus represent a new therapeutic approach to cancer therapy.

Sanfilippo syndrome type B (MPS IIIB) is a lysosomal storage disease caused by a deficiency of N-acetyl-glucosaminidase (NAGLU) activity. Since early therapeutic intervention is likely to yield the most efficacious results, we sought to determine the possible therapeutic utility of rAAV in early gene therapy based interventions. Currently, the application of recombinant adeno-associated virus (AAV) vectors is one of the most widely used gene transfer systems, and represents a promising approach in the treatment of MPS IIIB. From a translational standpoint, a minimally invasive, yet highly efficient method of vector administration is ideal. The thalamus is thought to be the switchboard for signal relay in the central nervous system (CNS) and therefore represents an attractive target. To identify an optimal AAV vector for early therapeutic intervention, and establish whether thalamic administration represents a feasible therapeutic approach, we performed a comprehensive assessment of transduction and biodistribution profiles of four green fluorescent protein (GFP) bearing rAAV serotypes, -5, -8, -9 and -rh10, administered bilaterally into the thalamus. Of the four serotypes compared, AAV8 and -9 proved superior to AAV5 and -rh10 both in biodistribution and transduction efficiency profiles. Genotype differences in transduction efficiency and biodistribution patterns were also observed. Importantly, we conclude that AAV8 and to a lesser extent, AAV9 represent preferable candidates for early gene therapy based intervention in the treatment of MPS IIIB. We also highlight the feasibility of thalamic rAAV administration, and conclude that this method results in moderate rAAV biodistribution with limited treatment capacity, thus suggesting a need for alternate methods of vector delivery.

12091 Background: Medical cannabis use is increasing significantly in the United States as states reduce restrictions. However, ambiguity concerning the evidence for medical cannabis efficacy and safety, especially in the field of oncology, is persistent. Clinicians therefore face challenges in examining benefits and risks of medical cannabis as adjuvant treatment for cancer patients. This study identifies and evaluates the most recent available evidence for the efficacy of cannabis and cannabinoids as adjuvant in supportive and/or palliative use in patients with cancer. Methods: Electronic databases searched included PubMed, Embase, Web of Science, and Cochrane Library to identify studies published following the latest available systematic review, between July 2016 through October 2019. Studies conducted outside the United States, studies not evaluating cannabis or cannabinoids in Oncology care, and preclinical studies were excluded. Findings were organized in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) framework. Lastly, qualitative synthesis was used to generate summary statements about the role of cannabis and cannabinoids as adjuvant in supportive and/or palliative cancer care. Results: We screened 2,267 articles and included 96 studies in our qualitative synthesis. Among those were 2 RCT’s (1 completed), 6 Systematic reviews with Meta-analysis, 4 Systematic reviews without Meta-analysis, 71 other types of reviews and 13 observational studies. The most frequently reported outcomes assessed were efficacy of cannabis and cannabinoids for: pain (40 of 96; 17 indicating improvement), nausea and vomiting (26 of 96; 20 indicating improvement), cachexia (22 of 96; 2 indicating improvement), and utilization patterns of cannabis and/or cannabinoids among cancer patients (8 of 96). Conclusions: Latest available prevalence estimates indicate that a significant proportion of patients in the United States with cancer use cannabis and/or cannabinoids (18.3-40.0%). There is substantial evidence for the effectiveness of cannabis and cannabinoids in treating cancer-related pain; specifically, oromucosal THC/CBD spray. There is conclusive evidence for the effectiveness of cannabis and cannabinoids in relieving chemotherapy-induced nausea and vomiting; specifically, oral THC. There is inconclusive evidence regarding the effectiveness of cannabis and cannabinoids in treating cancer-related cachexia.

The most aggressive and invasive tumor cells often reside in hypoxic microenvironments and rely heavily on rapid anaerobic glycolysis for energy production. This switch from oxidative phosphorylation to glycolysis, along with up-regulation of the glucose transport system, significantly increases the release of lactic acid from cells into the tumor microenvironment. Excess lactate and proton excretion exacerbate extracellular acidification to which cancer cells, but not normal cells, adapt. We have hypothesized that carbonic anhydrases (CAs) play a role in stabilizing both intracellular and extracellular pH to favor cancer progression and metastasis. Here we show that proton efflux (acidification) using the glycolytic rate assay is dependent on both extracellular pH (pHe) and CA IX expression. Yet, isoform selective sulfonamide-based inhibitors of CA IX did not alter proton flux, which suggests that the catalytic activity of CA IX is not necessary for this regulation. Other investigators have suggested the CA IX cooperates with the MCT transport family to excrete protons. To test this possibility, we examined the expression patterns of selected ion transporters and show that members of this family are differentially expressed within the molecular subtypes of breast cancer. The most aggressive form of breast cancer, triple negative breast cancer (TNBC), appears to coordinately express the monocarboxylate transporter 4 (MCT4) and carbonic anhydrase IX (CA IX). This supports a possible mechanism that utilizes the intramolecular H+shuttle system in CA IX to facilitate proton efflux through MCT4.

Carbonic anhydrase IX (CAIX) and XII (CAXII) are transmembrane proteins that are associated with cancer progression. We have previously described the catalytic properties of CAIX in MDA-MB-231 breast cancer cells, a line of cells that were derived from a patient with triple negative breast cancer. We chose this line because CAIX expression in breast cancer is a marker of hypoxia and a prognosticator for reduced survival. However, CAXII expression is associated with better survival statistics than those patients with low CAXII expression. Yet CAIX and CAXII have similar catalytic activities. Here we compare the potential roles of CAIX and CAXII in the context of TNBC and estrogen receptor (ER)-positive breast cancer. In tumor graft models, we show that CAIX and CAXII exhibit distinct expression patterns and non-overlapping. We find the same pattern across a panel of TNBC and luminal breast cancer cell lines. This affords an opportunity to compare directly CAIX and CAXII function. Our data suggest that CAIX expression is associated with growth potentiation in the tumor graft model and in a TNBC line using knockdown strategies and blocking activity with an impermeant sulfonamide inhibitor, N-3500. CAXII was not associated with growth potentiation. The catalytic activities of both CAIX and CAXII were sensitive to inhibition by N-3500 and activated at low pH. However, pH titration of activity in membrane ghosts revealed significant differences in the catalytic efficiency and pKa values. These features provide evidence that CAIX is a more efficient enzyme than CAXII at low pH and that CAIX shifts the equilibrium between CO2 and bicarbonate in favor of CO2 production by consuming protons. This suggests that in the acidic microenvironment of tumors, CAIX plays a role in stabilizing pH at a value that favors cancer cell survival.

Human cell lines are an important resource for research, and are often used as in vitro models of human diseases. In response to the mandate that all cells should be authenticated, we discovered that the MDA-MB-231 cells that were in use in our lab, did not validate based on the alleles of 9 different markers (STR Profile). We had been using this line as a model of triple negative breast cancer (TNBC) that has the ability to form tumors in immuno-compromised mice. Based on marker analysis, these cells most closely resembled the MCF10A line, which are a near diploid and normal mammary epithelial line. Yet, the original cells express carbonic anhydrase IX (CAIX) both constitutively and in response to hypoxia and are features that likely drive the aggressive nature of these cells. Thus, we sought to sub-purify CAIX-expressing cells using Fluorescence Activated Cell Sorting (FACS). These studies have revealed a new line of cells that we have name UFH-001, which have the TNBC phenotype, are positive for CAIX expression, both constitutively and in response to hypoxia, and behave aggressively in vivo. These cells may be useful for exploring mechanisms that underlie progression, migration, and metastasis of this phenotype. In addition, constitutive expression of CAIX allows its evaluation as a therapeutic target, both in vivo and in vitro.

Purpose The nonprofit Project Medishare launched a breast cancer treatment program in Port-au-Prince in July 2013 to address the demand for breast cancer care in Haiti. We outline the development of the program, highlight specific challenges, and discuss key considerations for others working in global oncology. Methods We reflected on our experiences in the key areas of developing partnerships, building laboratory capacity, conducting medical training, using treatment algorithms, and ensuring access to safe, low-cost chemotherapy drugs. We also critically reviewed our costs and quality measures. Results The program has treated a total of 139 patients with breast cancer with strong adherence to treatment regimens in 85% of patients. In 273 chemotherapy administrations, no serious exposure or adverse safety events were reported by staff. The mortality rate for 94 patients for whom we have complete data was 24% with a median survival time of 53 months. Our outcome data were likely influenced by stage at presentation, with more than half of patients presenting more than 12 months after first noticing a tumor. Future efforts will therefore focus on continuing to improve the level of care, while working with local partners to spread awareness, increase screening, and get more women into care earlier in the course of their disease. Conclusion Our experiences may inform others working to implement protocol-based cancer treatment programs in resource-poor settings and can provide valuable lessons learned for future global oncology efforts.

Sanfilippo syndrome type B (mucopolysaccharidosis IIIB, MPS IIIB) is a lysosomal storage disease resulting from deficiency of N-acetyl-glucosaminidase (NAGLU) activity. To determine the possible therapeutic utility of recombinant adeno-associated virus (rAAV) in early gene therapy-based interventions, we performed a comprehensive assessment of transduction and biodistribution profiles of four central nervous system (CNS) administered rAAV serotypes, -5, -8, -9 and -rh10. To simulate optimal earliest treatment of the disease, each rAAV serotype was injected into the CNS of neonatal MPS IIIB and control animals. We observed marked differences in biodistribution and transduction profiles between the serotypes and this differed in MPS IIIB compared with healthy control mice. Overall, in control mice, all serotypes performed comparably, although some differences were observed in certain focal areas. In MPS IIIB mice, AAV8 was more efficient than AAV5, -9 and -rh10 for gene delivery to most structures analyzed, including the cerebral cortex, hippocampus and thalamus. Noteworthy, the pattern of biodistribution within the CNS varied by serotype and genotype. Interestingly, AAV8 also produced the highest green fluorescent protein intensity levels compared with any other serotype and demonstrated improved transduction in NAGLU compared with control brains. Importantly, we also show leakage of AAV8, -9 and -rh10, but not AAV5, from CNS parenchyma to systemic organs. Overall, our data suggest that AAV8 represents the best therapeutic gene transfer vector for early intervention in MPS IIIB.

Purpose: To evaluate the feasibility of functional photoacoustic tomography (fPAT) for high resolution detection and characterization of breast cancer and to demonstrate for the first time quantitative hemoglobin concentration and oxygen saturation images of breasts that were formed with model-based reconstruction of tomographic photoacoustic data. Methods: The study was HIPAA compliant and was approved by the university institutional review board. Written informed consents were obtained from all the participants. Ten cases, including six cancer and four healthy (mean age = 50 yr; age range = 41–66 yr), were examined. Functional images of breast tissue including absolute total hemoglobin concentration (HbT) and oxygen saturation (StO2%) were obtained by fPAT and cross validated with magnetic resonance imaging (MRI) readings and/or histopathology. Results: HbT and StO2% maps from all six pathology-confirmed cancer cases (60%) show clear detection of tumor, while MR images indicate clear detection of tumor for five of six cancer cases; one small tumor was read as near-complete-resolution by MRI. The average HbT and StO2% value of suspicious lesion area for the cancer cases was 61.6 ± 18.9 μM/l and 67.5% ± 5.2% compared to 25.6 ± 7.4 μM/l and 65.2% ± 3.8% for background normal tissue. Conclusions: fPAT has the potential to be a significant add-on in breast cancer detection and characterization as it provides submillimeter resolution functional images of breast lesions.

Background: The taxanes and platinum agents provide substantial improvement in the treatment of breast and other cancers. Neuropathy is often the dose limiting toxicity of these agents and can devastatingly affect the patient due to diminished fine motor skills and pain in the hands and feet that diminishes the ability to exercise and interact in normal life. Clinically significant neuropathy occurs in 40% of patients and >10% will persist past a year causing permanent effects on quality of life. Perhaps more devastating, patients often have to discontinue effective treatment due to the development of these symptoms. Additionally, anemia contributes to dose reductions and delays that compromise therapy and cause significant fatigue that diminishes quality of life. Interventions that could reduce these symptoms would provide a substantial improvement in the ability to care for these patients. Toward this end we have developed a non-toxic dietary approach that incorporates supplementation with several natural products. Together, this therapeutic approach, called CS.001 is able to alleviate many of the negative side effects of breast cancer chemotherapy. Methods: C57BL/6 mice were treated with Paclitaxel weekly or Oxaliplatin three times weekly. Animals were tested for neuropathic pain using a cold sensitivity test [acetone test] and sensitivity to mechanical stimulus [Von Frey Test]. A CBC and CMP were performed to assess systemic toxicity. Animals were provided with a nutritional complete diet that limited their carbohydrates to 10% of total caloric input and were additionally supplemented with the following natural products: [1] Medium chain triglycerides [30g/kg], [2] Curcumin [1200mg/kg], [3]EGCG [1200mg/kg] and [4]broccoli sprout powder [20g/kg]. Animals were placed on the CS.001 diet 1 week before beginning chemotherapy. Results: Paclitaxel & Oxaliplatin treatment resulted in a statistically significantly increase in sensitivity to cold stimulus [Acetone test, p Conclusion: Dietary intervention combined with a supplementation of 4 natural products was able to attenuate chemotherapy induced anemia and neuropathy following chemotherapy treatment in mice. This combination is a promising quality of life intervention to evaluate in cancer patients receiving taxanes or platinums. Citation Format: Coy D Heldermon, Ben G Griffith, Matt Bloom, Jennifer Owen, Regina J Martuscello, Yosef Schwartz, Brent A Reynolds, Loic P Deleyrolle. A combination natural product therapy attenuates common side effects associated with chemotherapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-15-10.

EGFR, HER2, and HER3 contribute to the initiation and progression of human cancers, and are therapeutic targets for monoclonal antibodies and tyrosine kinase inhibitors. An important source of resistance to these agents arises from functional redundancy among EGFR, HER2, and HER3. EGFR family members contain conserved extracellular structures that are stabilized by disulfide bonds. Compounds that disrupt extracellular disulfide bonds could inactivate EGFR, HER2, and HER3 in unison. Here we describe the identification of compounds that kill breast cancer cells that overexpress EGFR or HER2. Cell death parallels downregulation of EGFR, HER2, and HER3. These compounds disrupt disulfide bonds and are termed Disulfide Bond Disrupting Agents (DDAs). DDA RBF3 exhibits anticancer efficacy in vivo at 40 mg/kg without evidence of toxicity. DDAs may complement existing EGFR-, HER2-, and HER3-targeted agents that function through alternate mechanisms of action, and combination regimens with these existing drugs may overcome therapeutic resistance.

Purpose Little is known about the epidemiology of breast cancer in developing countries, and Haiti has perhaps the least data of any country in the Western Hemisphere. Methods We conducted a retrospective review of all patients enrolled in an ongoing breast cancer treatment program in Port-au-Prince, Haiti, from July 1, 2013, through June 30, 2017. Data were drawn from each patient's electronic medical record, paper chart, and biopsy results. Results The records of 525 women with breast cancer were reviewed for this study. The median age at presentation was 49 years (n = 507). The risk factors observed were as follows: postmenopausal, 50.8% (n = 354); nulliparity, 15.7% (n = 338); hormonal contraception use, 35.0% (n = 309); never breastfed, 20.6% (n = 316); family history of any cancer, 22.0% (n = 295); overweight, 51.5% (n = 332); and smoking, 5.0% (n = 338). Of all those staged, 83.9% (n = 447) of the patients presented with stage III/IV disease and more than half delayed care for > 12 months after first noticing a breast mass. For the subset of tumors for which estrogen receptor (ER; n = 245) and human epidermal growth factor receptor 2 (HER2; n = 179) status was available, the prevalence of ER-positive tumors was 51.8%, of HER2-positive tumors was 19.6%, and of triple-negative tumors was 38.5%. The 12-month mortality rate (n = 425) was 18.4% overall and 27.5% for those who presented with stage IV disease. Median survival was not reached. Conclusion Breast cancer in Haiti presents at an early age and advanced stage. Triple-negative, ER-negative, and high-grade tumors are common. Delays in seeking care and incomplete treatment likely contribute to the high mortality rate; however, as in black women in the United States, the distribution of tumor types may contribute to disparate outcomes.

Treatment with lapatinib results in many toxicities. Described here is a novel toxicity, mucosal discoloration that developed after lapatinib was initiated in a breast cancer patient.

Despite much progress, the prognosis for patients with relapsed or refractory multiple myeloma (RRMM) remains poor. Novel therapies including the immunomodulatory agents and proteasome inhibitors h...

Mucopolysaccharidosis type IIIB (MPS IIIB) or Sanfilippo Syndrome type B is a lysosomal storage disease resulting from the deficiency of N-acetyl glucosaminidase (NAGLU) activity. We previously showed that intracranial adeno-associated virus (AAV)-based gene therapy results in partial improvements of several aspects of the disease. In an attempt to further correct the disease, MPS IIIB mice were treated at 2-4 days of age with intracranial AAV2/5-NAGLU (IC-AAV), intravenous lentiviral-NAGLU (IV-LENTI) or the combination of both (BOTH). The BOTH group had the most complete biochemical and histological improvements of any treatment group. Compared with untreated MPS IIIB animals, all treatments resulted in significant improvements in motor function (rotarod) and hearing (auditory-evoked brainstem response). In addition, each treatment group had a significantly increased median life span compared with the untreated group (322 days). The combination arm had the greatest increase (612 days), followed by IC-AAV (463 days) and IV-LENTI (358 days). Finally, the BOTH group had nearly normal circadian rhythm measures with improvement in time to activity onset. In summary, targeting both the systemic and central nervous system disease of MPS IIIB early in life appears to be the most efficacious approach for this inherited metabolic disorder.

Carcinoma cells can acquire increased motility and invasiveness through epithelial-to-mesenchymal transition (EMT). However, the significance of EMT in cancer metastasis has been controversial, and the exact fates and functions of EMT cancer cells in vivo remain inadequately understood. Here, we tracked epithelial cancer cells that underwent inducible or spontaneous EMT in various tumor transplantation models. Unlike epithelial cells, the majority of EMT cancer cells were specifically located in the perivascular space and closely associated with blood vessels. EMT markedly activated multiple pericyte markers in carcinoma cells, in particular PDGFR-β and N-cadherin, which enabled EMT cells to be chemoattracted towards and physically interact with endothelium. In tumor xenografts generated from carcinoma cells that were prone to spontaneous EMT, a substantial fraction of the pericytes associated with tumor vasculature were derived from EMT cancer cells. Depletion of such EMT cells in transplanted tumors diminished pericyte coverage, impaired vascular integrity, and attenuated tumor growth. These findings suggest that EMT confers key pericyte attributes on cancer cells. The resulting EMT cells phenotypically and functionally resemble pericytes and are indispensable for vascular stabilization and sustained tumor growth. This study thus proposes a previously unrecognized role for EMT in cancer.

// Renan B. Ferreira 1, * , Mengxiong Wang 2, 3, * , Mary E. Law 2, 3 , Bradley J. Davis 2, 3 , Ashton N. Bartley 1 , Paul J. Higgins 4 , Michael S. Kilberg 5 , Katherine E. Santostefano 6 , Naohiro Terada 6 , Coy D. Heldermon 7 , Ronald K. Castellano 1 , Brian K. Law 2, 3 1 Department of Chemistry, University of Florida, Gainesville, FL 32611, USA 2 Department of Pharmacology & Therapeutics, University of Florida, Gainesville, FL 32610, USA 3 UF-Health Cancer Center, University of Florida, Gainesville, FL 32610, USA 4 Center for Cell Biology and Cancer Research, Albany Medical College, Albany, NY 12208, USA 5 Department of Biochemistry, University of Florida, Gainesville, FL, 32610, USA 6 Department of Pathology, Immunology, and Laboratory Medicine, Center for Cellular Reprogramming, University of Florida College of Medicine, Gainesville, FL 32610, USA 7 Department of Medicine, University of Florida, Gainesville, FL, 32610, USA * These authors have contributed equally to this work Correspondence to: Brian K. Law, email: bklaw@ufl.edu Ronald K. Castellano, email: castellano@chem.ufl.edu Keywords: HER2, EGFR, UPR, Akt, breast cancer Received: August 15, 2016 Accepted: February 12, 2017 Published: March 07, 2017 ABSTRACT Many breast cancer deaths result from tumors acquiring resistance to available therapies. Thus, new therapeutic agents are needed for targeting drug-resistant breast cancers. Drug-refractory breast cancers include HER2+ tumors that have acquired resistance to HER2-targeted antibodies and kinase inhibitors, and “Triple-Negative” Breast Cancers (TNBCs) that lack the therapeutic targets Estrogen Receptor, Progesterone Receptor, and HER2. A significant fraction of TNBCs overexpress the HER2 family member Epidermal Growth Factor Receptor (EGFR). Thus agents that selectively kill EGFR+ and HER2+ tumors would provide new options for breast cancer therapy. We previously identified a class of compounds we termed Disulfide bond Disrupting Agents (DDAs) that selectively kill EGFR+ and HER2+ breast cancer cells in vitro and blocked the growth of HER2+ breast tumors in an animal model. DDA-dependent cytotoxicity was found to correlate with downregulation of HER1-3 and Akt dephosphorylation. Here we demonstrate that DDAs activate the Unfolded Protein Response (UPR) and that this plays a role in their ability to kill EGFR+ and HER2+ cancer cells. The use of breast cancer cell lines ectopically expressing EGFR or HER2 and pharmacological probes of UPR revealed all three DDA responses: HER1-3 downregulation, Akt dephosphorylation, and UPR activation, contribute to DDA-mediated cytotoxicity. Significantly, EGFR overexpression potentiates each of these responses. Combination studies with DDAs suggest that they may be complementary with EGFR/HER2-specific receptor tyrosine kinase inhibitors and mTORC1 inhibitors to overcome drug resistance.