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Treatment of advanced breast cancer remains challenging. Copper and some of the copper-dependent proteins are emerging therapeutic targets because they are essential for cell proliferation and survival, and have been shown to stimulate angiogenesis and metastasis. Here, we show that DCAC50, a recently developed small-molecule inhibitor of the intracellular copper chaperones, ATOX1 and CCS, reduces cell proliferation and elevates oxidative stress, triggering apoptosis in a panel of triple-negative breast cancer (TNBC) cells. Inhibition of ATOX1 activity with DCAC50 disrupts copper homeostasis, leading to increased copper levels, altered spatial copper redistribution, and accumulation of ATP7B to the cellular perinuclear region. The extent and impact of this disruption to copper homeostasis vary across cell lines and correlate with cellular baseline copper and glutathione levels. Ultimately, treatment with DCAC50 attenuates tumor growth and suppresses angiogenesis in a xenograft mouse model, and prevents endothelial cell network formation in vitro. Co-treatment with paclitaxel and DCAC50 enhances cytotoxicity in TNBC and results in favorable dose reduction of both drugs. These data demonstrate that inhibition of intracellular copper transport targets tumor cells and the tumor microenvironment, and is a promising approach to treat breast cancer.

Figure S1 - Western blot images showing ATOX1 and CCS levels in TNBC cells, corresponding to data presented in Figure 1. Figure S2 - Dose-response profiles of TM in TNBC cells and DCAC50 in HMEC; caspase-3/7 activity after DCAC50 treatment of HMEC. Figure S3 - Copper content, copper distribution and iron and zinc content for DCAC50-treated TNBC cells, supplementing Figure 3. Figure S4 - Protein levels of copper transporters, GSH levels, GSH depletion experiments and SOD1 activity levels in TNBC cells in response to DCAC50 treatment, supplementing Figure 4. Figure S5 - Dose-response profile for DCAC50 and caspase-3/7 activity after DCAC50 treatment in HuVEC. Figure S6 - Evaluation of DCAC50 treatment on tumor volumes and angiogenesis in MDA-MB-468 and MDA-MB-231 xenograft mouse models, supplementing Figure 5. Supplementary Methods ¬- Information about the antibodies and detailed procedures used for Western blot, immunofluorescent staining and SOD activity assay.

Background: Life-threatening immune-related adverse events (irAEs) that require hospital admission are not uncommon in patients treated with immune checkpoint inhibitors (ICIs). The clinical and hematological parameters are attractive biomarkers as potential predictors of irAE. Methods: This is a retrospective study of patients with melanoma and lung cancer treated with ICIs between 2015 and 2019 at the University of South Alabama Mitchell Cancer Institute. Fisher’s exact test, Pearson Chi-squared test, log-rank test, and Cox proportional hazard model were used to evaluate clinical and hematological parameters as possible predictors of irAE. Results: The cohort consisted of 160 patients treated with at least two doses of ICI, of which 54 (33.8%) patients had melanoma and 106 (66.3%) had lung cancer. Incidence of irAE did not have any bearing on the overall survival (OS) or progression-free survival (PFS) of the cohort. The clinical factors associated with irAE were dual-agent therapy (ipilimumab/nivolumab combination) and high disease burden (? 2 metastatic sites). The irAE-group had a lower mean platelet-to-lymphocyte ration (PLR, 200 vs. 257, P = 0.04). Although not statistically significant at the level of 0.05, other factors such as type of cancer (lung cancer > melanoma (P = 0.06)), stage at treatment (stage IV > stage II and III disease (P = 0.06)), and higher absolute lymphocyte counts (P = 0.07) showed a considerable association with irAE and warrants further review with different patient data. Conclusions: Irrespective of ICI used to treat lung cancer and melanoma, patients with high disease burden and dual-agent ICI therapy were more prone to irAE. The only hematological parameter that may predict the incidence of irAE is low baseline PLR. J Clin Med Res. 2021;13(5):268-275 doi: https://doi.org/10.14740/jocmr4511

Bruton tyrosine kinase (BTK) inhibitors have become an important therapy for untreated and previously treated patients with chronic lymphocytic leukemia (CLL). Despite improved outcomes, rare adverse events, such as invasive fungal infections, have been reported with the use of first-generation BTK inhibitors. Invasive fungal infections carry a high morbidity and mortality risk. There have been several case reports describing the association between aspergillosis and ibrutinib treatment, but none with acalabrutinib, to our knowledge. In this case report, we describe a patient with CLL who developed an intracranial Aspergillus fumigatus infection while receiving acalabrutinib.

Based on the literature review, many studies have been inconclusive in regards to adenoma detection and procedural positioning during a colonoscopy. Scope looping can make cecal intubation challenging, changing the positioning of the patient and application of external abdominal pressure can overcome this difficulty. A colonoscopy in a prone position can overcome these challenges and reduce cecal intubation time. It can thus improve the safety of the patient and the staff by minimizing the movement of a sedated patient.

e15159 Background: Immune-related adverse events (irAE) remain a significant challenge with the expansion of checkpoint inhibitors (ICI) indications. Unlike previous studies published, we investigated risk factors for irAE development, including lymphocytes and neutrophils counts in lung cancer and melanoma treated with all available ICIs in current clinical practice. Methods: This is a retrospective study conducted at the University of South Alabama Mitchell Cancer Institute. Between 2015-2019. A total of 160 patients with a diagnosis of melanoma (N = 54) or lung cancer (N = 106) who received at least two doses of ICI including ipilimumab (15%), nivolumab (32%), pembrolizumab (35%), dual nivolumab/ipilimumab (5%), durvalumab (9%) and atezolizumab (4%). The patient's baseline characteristics were extracted with irAE (grade 3/4) details and survival outcomes. Descriptive statistics were used, Fisher exact test to compare categorical variables, and Wilcoxon rank sum test for continuous variables using JMP software. Results: The median age at diagnosis was 64 years (range 17-93), with 51% females. Race distribution with 76% Caucasians and 26% African Americans. Around 30% of the cohort was treated for recurrence, and 39% did receive prior systemic chemotherapy. Median overall survival (OS) was 13.5 months (m) for melanoma and 16 m for lung cancer with CI 95% [16-24] and [15-23], respectively. Twenty-nine (29%) percent of the cohort (N = 46) had grade 3/4 irAEs. Median of baseline hematological parameters including total white blood count (WBC), absolute neutrophil count (ANC), absolute lymphocyte count (ALC), ANC to ALC ratio, and platelet to ALC ratio of these patients were not statistically different from the cohort without grade 3/4 irAEs. Interestingly, if a patient has baseline ALC < 1K/μL, the risk of irAE recurrence is low when ICI is re-initiated, p = .0143 (after symptomatic recovery from irAEs). Conclusions: Irrespective of ICI used, baseline lymphocyte count, and its relation to other blood counts have no clear impact on irAE. Larger cohorts or prospective studies are needed to make stronger conclusions about the relationship between the immune system and the occurrence of irAEs

Treatment of advanced breast cancer remains challenging. Copper and some of the copper-dependent proteins are emerging therapeutic targets because they are essential for cell proliferation and survival, and have been shown to stimulate angiogenesis and metastasis. Here, we show that DCAC50, a recently developed small-molecule inhibitor of the intracellular copper chaperones, ATOX1 and CCS, reduces cell proliferation and elevates oxidative stress, triggering apoptosis in a panel of triple-negative breast cancer (TNBC) cells. Inhibition of ATOX1 activity with DCAC50 disrupts copper homeostasis, leading to increased copper levels, altered spatial copper redistribution, and accumulation of ATP7B to the cellular perinuclear region. The extent and impact of this disruption to copper homeostasis vary across cell lines and correlate with cellular baseline copper and glutathione levels. Ultimately, treatment with DCAC50 attenuates tumor growth and suppresses angiogenesis in a xenograft mouse model, and prevents endothelial cell network formation in vitro. Co-treatment with paclitaxel and DCAC50 enhances cytotoxicity in TNBC and results in favorable dose reduction of both drugs. These data demonstrate that inhibition of intracellular copper transport targets tumor cells and the tumor microenvironment, and is a promising approach to treat breast cancer.

Introduction: Non-small cell lung cancer (NSCLC) is the top cause of cancer-related mortality. We have identified a role for the tumor suppressor hepatocyte growth factor activator inhibitor type-1 (HAI-1) in human lung adenocarcinoma. HAI-1 loss results in unregulated downstream MET/RON tyrosine kinase receptor signaling. We hypothesized that HAI-1 is lost in human lung adenocarcinomas and could serve as a biomarker for therapy with crizotinib, a tyrosine kinase inhibitor (TKI) with activity against both MET and RON. We also hypothesized that unregulated RON signaling in the tumor microenvironment, via HAI-1 loss, may promote tumor metastasis by skewing tumor associated macrophages (TAMs) from an anti-tumor (M1) to a pro-tumor (M2) phenotype. Methods: Immunohistochemical staining with HAI-1 antibody (1N7) was performed on human tissue microarray containing 20 normal and 20 lung tumor specimens. Intensity grading (0, 1+, 2+, 3+) was performed, and percent cell expression was calculated for each group. For all in vitro assays, H358 lung adenocarcinoma cells, which express high levels of endogenous HAI-1, were stably transfected with mock shRNA or HAI-1 shRNA. Cell protein expression and phosphorylation was assessed via western blot. Cell viability after exposure to crizotinib was assessed by trypan blue staining. For flow cytometric analysis of macrophage phenotype, undifferentiated (M0) macrophages were exposed to conditioned medium from transfected H358 cells and macrophage phenotype was assessed by flow for surface markers CD68 (M0), CCR7 (M1), and CD206 (M2). Results: HAI-1 expression is significantly decreased in human lung adenocarcinoma compared to normal lung with overall HAI-1 IHC positivity ~20% compared to ~60% respectively. HAI-1 knockdown in H358 cells caused increased phosphorylation of RON and increased sensitivity to crizotinib. Culturing M0 macrophages in conditioned media from H358 shHAI-1 knockdown cells caused a decrease in the CCR7 positive M1 fraction from 8.4% to 1.9%, and an increase in the CD206 positive M2 fraction from 0.77% to 2.09%. Conclusion: We have demonstrated that loss of HAI-1 occurs in human lung adenocarcinoma consistent with previous studies in human NSCLC cell lines. Furthermore, we showed significantly increased RON signaling activity after HAI-1 knockdown. We have also demonstrated that HAI-1 loss can skew tumor associated macrophages (TAMs) from an anti-tumor (M1) to a pro-tumor (M2) phenotype. We have shown that loss of HAI-1 in vitro can increase sensitivity to crizotinib, suggesting that loss of HAI-1 may predict for tumor sensitivity to MET/RON inhibition. Our findings show that dysregulation of the HAI-1/MET/RON pathway exerts effects both on lung tumor cells as well as TAMs, making this pathway a potentially powerful therapeutic target. Citation Format: Austin J. McHenry, Gautam Sondarva, Vijayalakshmi Ananthanarayanan, Ashley Hess, Patricia E. Simms, Alhareth Alsayed, Hiroaki Kataoka, Jan Marusarz, Sandeep Kumar, Ravi Salgia, Arkadiusz Dudek, Stanley Borowicz, Ajay Rana. Loss of hepatocyte growth factor activator inhibitor type-1 (HAI-1) in human lung adenocarcinomas promotes RON receptor phosphorylation and increased sensitivity to crizotinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5514.

The Forkhead box M1 (FOXM1) is a transcription factor that has been implicated in normal cell growth and proliferation through control of cell cycle transition and mitotic spindle. It is implicated in carcinogenesis of various malignancies where it is activated by either amplification, increased stability, enhanced transcription, dysfunction of regulatory pathways, or activation of PI3K/AKT, epidermal growth factor receptor, Raf/MEK/MAPK, and Hedgehog pathways. This review describes the role of FOXM1 in breast cancer. This includes how FOXM1 impacts on different subtypes of breast cancer, that is, luminal/estrogen receptor positive (ER+), expressing human epidermal growth factor receptor 2 (HER2), basal-like breast cancer (BBC), and triple negative breast cancer (TNBC). The review also describes different tested preclinical therapeutic strategies targeting FOXM1. Developing clinically applicable therapies that specifically inhibit FOXM1 activity is a logical next step in biomarker-driven approaches against breast cancer but will not be without its challenges due to the unique properties of this transcription factor.

Rationale: Treatment of patients with triple-negative breast cancer (TNBC) remains challenging due to unpredictable disease progression and acquired resistance to chemotherapy. Finding novel targets in TNBC is important for the development of anticancer therapy. Cancer cells frequently adapt to cytotoxicity of existing pharmacological agents by exploiting metabolic pathways. Elevated copper metabolism and adaptations to oxidative stress have been linked to cancer progression. Copper chelation with tetrathiomolybdate (TM) was reported to control disease progression, especially in patients with TNBC. However, long-term consequences of global copper depletion are still under investigation. An alternative strategy is provided by a recently developed small molecule DCAC50 that blocks the copper transfer interface of two major copper chaperons, ATOX-1 and CCS. Protein levels of ATOX-1 and CCS are elevated in TNBC cells compared to normal cells. Thus, we hypothesized that disrupting copper transport by targeting ATOX-1 and CCS with DCAC50 may suppress TNBC progression. Results: We investigated efficacy and potency of novel small molecule DCAC50 to induce cytotoxicity in a panel of TNBC cells lines. DCAC50 reduced cell proliferation in dose-dependent manner, measured by MTS assay. IC50 doses ranged from 3-10uM. Most importantly, DCAC50 induced apoptosis in TNBC cells, detected by Caspase 3/7 activity and Annexin V/PI staining. As expected, DCAC50 elevated intracellular copper levels. Moreover, TNBC cells treated with DCAC50 had significantly higher levels of oxidized glutathione (GSSG) and caused increased oxidation of DCF-DA reagent, demonstrating elevated oxidative stress. Surprisingly, activity of CCS downstream target, SOD1, was not affected by selective inhibition of copper transport with DCAC50; suggesting that intracellular copper accumulation and oxidative stress is mediated by ATOX-1 inhibition. Interestingly, copper depletion with TM inhibited SOD1 activity but failed to significantly impact oxidative stress in TNBC cells. Recognizing DCAC50 generates oxidative stress triggering apoptosis in TNBC cells we evaluated benefits of combining DCAC50 treatment with paclitaxel. Multi-drug combination dose-response analysis revealed that co-treatment induced synergistic cytotoxicity and resulted in favorable dose reduction of both drugs. Conclusions: Selective inhibition of copper metabolism with novel small molecule DCAC50 elevates oxidative stress triggering apoptosis in TNBC cells. This approach may be valuable in combination with chemotherapy especially when tumor cells acquire resistance to first line therapy in TNBC. Ongoing studies comparing selective inhibition of copper transport to global copper chelation, and investigating efficacy and biological activity of DCAC50 in vivo will help further estimate benefits and clinical relevance of this approach for treatment of TNBC patients. Citation Format: Olga Karginova, Claire Weekley, Akila Raoul, Alhareth Alsayed, Tong Wu, Chuan He, Olufunmilayo I. Olopade. Selective inhibition of copper metabolism as a novel approach to treat triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3552. doi:10.1158/1538-7445.AM2017-3552