Evaluation of Copanlisib in Combination with Eribulin in Triple-negative Breast Cancer Patient-derived Xenograft Models.

The PI3K pathway regulates essential cellular functions and promotes chemotherapy resistance. Activation of PI3K pathway signaling is commonly observed in triple-negative breast cancer (TNBC). However previous studies that combined PI3K pathway inhibitors with taxane regimens have yielded inconsistent results. We therefore set out to examine whether the combination of copanlisib, a clinical grade pan-PI3K inhibitor, and eribulin, an antimitotic chemotherapy approved for taxane-resistant metastatic breast cancer, improves the antitumor effect in TNBC. A panel of eight TNBC patient-derived xenograft (PDX) models was tested for tumor growth response to copanlisib and eribulin, alone or in combination. Treatment-induced signaling changes were examined by reverse phase protein array, immunohistochemistry (IHC) and 18F-fluorodeoxyglucose PET (18F-FDG PET). Compared with each drug alone, the combination of eribulin and copanlisib led to enhanced tumor growth inhibition, which was observed in both eribulin-sensitive and -resistant TNBC PDX models, regardless of PI3K pathway alterations or PTEN status. Copanlisib reduced PI3K signaling and enhanced eribulin-induced mitotic arrest. The combination enhanced induction of apoptosis compared with each drug alone. Interestingly, eribulin upregulated PI3K pathway signaling in PDX tumors, as demonstrated by increased tracer uptake by 18F-FDG PET scan and AKT phosphorylation by IHC. These changes were inhibited by the addition of copanlisib. These data support further clinical development for the combination of copanlisib and eribulin and led to a phase I/II trial of copanlisib and eribulin in patients with metastatic TNBC. SIGNIFICANCE: In this research, we demonstrated that the pan-PI3K inhibitor copanlisib enhanced the cytotoxicity of eribulin in a panel of TNBC PDX models. The improved tumor growth inhibition was irrespective of PI3K pathway alteration and was corroborated by the enhanced mitotic arrest and apoptotic induction observed in PDX tumors after combination therapy compared with each drug alone. These data provide the preclinical rationale for the clinical testing in TNBC.

Oncogenic Gq/11 signaling acutely drives and chronically sustains metabolic reprogramming in uveal melanoma.

Metabolic reprogramming has been shown to occur in uveal melanoma (UM), the most common intraocular tumor in adults. Mechanisms driving metabolic reprogramming in UM are poorly understood. Elucidation of these mechanisms could inform development of new therapeutic strategies for metastatic UM, which has poor prognosis because existing therapies are ineffective. Here, we determined whether metabolic reprogramming is driven by constitutively active mutant α-subunits of the heterotrimeric G proteins Gq or G11 (Gq/11), the oncogenic drivers in ∼90% of UM patients. Using PET-computed tomography imaging, microphysiometry, and GC/MS, we found that inhibition of oncogenic Gq/11 with the small molecule FR900359 (FR) attenuated glucose uptake by UM cells in vivo and in vitro, blunted glycolysis and mitochondrial respiration in UM cell lines and tumor cells isolated from patients, and reduced levels of several glycolytic and tricarboxylic acid cycle intermediates. FR acutely inhibited glycolysis and respiration and chronically attenuated expression of genes in both metabolic processes. UM therefore differs from other melanomas that exhibit a classic Warburg effect. Metabolic reprogramming in UM cell lines and patient samples involved protein kinase C and extracellular signal-regulated protein kinase 1/2 signaling downstream of oncogenic Gq/11. Chronic administration of FR upregulated expression of genes involved in metabolite scavenging and redox homeostasis, potentially as an adaptive mechanism explaining why FR does not efficiently kill UM tumor cells or regress UM tumor xenografts. These results establish that oncogenic Gq/11 signaling is a crucial driver of metabolic reprogramming in UM and lay a foundation for studies aimed at targeting metabolic reprogramming for therapeutic development.

In vivo quantitative assessment of therapeutic response to bortezomib therapy in disseminated animal models of multiple myeloma with [18F]FDG and [64Cu]Cu-LLP2A PET.

BACKGROUND: Multiple myeloma (MM) is a disease of cancerous plasma cells in the bone marrow. Imaging-based timely determination of therapeutic response is critical for improving outcomes in MM patients. Very late antigen-4 (VLA4, CD49d/CD29) is overexpressed in MM cells. Here, we evaluated [18F]FDG and VLA4 targeted [64Cu]Cu-LLP2A for quantitative PET imaging in disseminated MM models of variable VLA4 expression, following bortezomib therapy. METHODS: In vitro and ex vivo VLA4 expression was evaluated by flow cytometry. Human MM cells, MM.1S-CG and U266-CG (C: luciferase and G: green fluorescent protein), were injected intravenously in NOD-SCID gamma mice. Tumor progression was monitored by bioluminescence imaging (BLI). Treatment group received bortezomib (1 mg/kg, twice/week) intraperitoneally. All cohorts (treated, untreated and no tumor) were longitudinally imaged with [18F]FDG (7.4-8.0 MBq) and [64Cu]Cu-LLP2A (2-3 MBq; Molar Activity: 44.14 ± 1.40 MBq/nmol) PET, respectively. RESULTS: Flow cytometry confirmed high expression of CD49d in U266 cells (> 99%) and moderate expression in MM.1S cells (~ 52%). BLI showed decrease in total body flux in treated mice. In MM.1S-CG untreated versus treated mice, [64Cu]Cu-LLP2A localized with a significantly higher SUVmean in spine (0.58 versus 0.31, p < 0.01) and femur (0.72 versus 0.39, p < 0.05) at week 4 post-tumor inoculation. There was a four-fold higher uptake of [64Cu]Cu-LLP2A (SUVmean) in untreated U266-CG mice compared to treated mice at 3 weeks post-treatment. Compared to [64Cu]Cu-LLP2A, [18F]FDG PET detected treatment-related changes at later time points. CONCLUSION: [64Cu]Cu-LLP2A is a promising tracer for timely in vivo assessment of therapeutic response in disseminated models of MM.

Mapping nephron mass in vivo using positron emission tomography.

Nephron number varies widely in humans. A low nephron endowment at birth or a loss of functioning nephrons is strongly linked to increased susceptibility to chronic kidney disease. In this work, we developed a contrast agent, radiolabeled cationic ferritin (RadioCF), to map functioning glomeruli in vivo in the kidney using positron emission tomography (PET). PET radiotracers can be detected in trace doses (<30 nmol), making them useful for rapid clinical translation. RadioCF is formed from cationic ferritin (CF) and with a radioisotope, Cu-64, incorporated into the ferritin core. We showed that RadioCF binds specifically to kidney glomeruli after intravenous injection in mice, whereas radiolabeled noncationic ferritin (RadioNF) and free Cu-64 do not. We then showed that RadioCF-PET can distinguish kidneys in healthy wild-type (WT) mice from kidneys in mice with oligosyndactylism (Os/+), a model of congenital hypoplasia and low nephron mass. The average standardized uptake value (SUV) measured by PET 90 min after injection was 21% higher in WT mice than in Os/+ mice, consistent with the higher glomerular density in WT mice. The difference in peak SUV from SUV at 90 min correlated with glomerular density in male mice from both WT and Os/+ cohorts (R2 = 0.98). Finally, we used RadioCF-PET to map functioning glomeruli in a donated human kidney. SUV within the kidney correlated with glomerular number (R2= 0.78) measured by CF-enhanced magnetic resonance imaging in the same locations. This work suggests that RadioCF-PET appears to accurately detect nephron mass and has the potential for clinical translation.