Dermal Delivery of Constructs Encoding Cre Recombinase to Induce Skin Tumors in PtenLoxP/LoxP;BrafCA/+ Mice

Current genetically-engineered mouse melanoma models are often based on Tyr::CreERT2-controlled MAPK pathway activation by the BRAFV600E mutation and PI3K pathway activation by loss of PTEN. The major drawback of these models is the occurrence of spontaneous tumors caused by leakiness of the Tyr::CreERT2 system, hampering long-term experiments. To address this problem, we investigated several approaches to optimally provide local delivery of Cre recombinase, including injection of lentiviral particles, DNA tattoo administration and particle-mediated gene transfer, to induce melanomas in PtenLoxP/LoxP;BrafCA/+ mice lacking the Tyr::CreERT2 allele. We found that dermal delivery of the Cre recombinase gene under the control of a non-specific CAG promoter induced the formation of melanomas, but also keratoacanthoma and squamous cell carcinomas. Delivery of Cre recombinase DNA under the control of melanocyte-specific promoters in PtenLoxP/LoxP;BrafCA/+ mice resulted in sole melanoma induction. The growth rate and histological features of the induced tumors were similar to 4-hydroxytamoxifen-induced tumors in Tyr::CreERT2;PtenLoxP/LoxP;BrafCA/+ mice, while the onset of spontaneous tumors was prevented completely. These novel induction methods will allow long-term experiments in mouse models of skin malignancies

Targeting Estrogen Receptor Beta in a Phase 2 Study of High-Dose Estradiol in Metastatic Triple-Negative Breast Cancer: A Wisconsin Oncology Network Study

BACKGROUND: Estrogen receptor beta (ERβ) is expressed by 50% to 80% of triple-negative breast cancers (TNBC). Agonism of ERβ has antiproliferative effects in TNBC cells expressing ERβ. This phase 2 study evaluated single-agent high-dose estradiol in patients with advanced TNBC. PATIENTS AND METHODS: Adult women with measurable advanced TNBC were treated with estradiol 10 mg oral 3 times daily provided continuously for 28-day cycles. A Simon optimal 2-stage design was used. The primary end point was objective response (OR). Secondary end points included progression-free survival (PFS), clinical benefit (CB), and safety. OR, CB, and PFS by ERβ status were also examined. RESULTS: Seventeen evaluable women were enrolled. Median age was 58 years (range, 34-90 years); the median number of prior systemic therapies was 2 (range, 0-6). One patient had a confirmed partial response (OR rate, 5.9%) and remained on the study for \u3e 24 weeks. Three patients had stable disease, with one lasting more than 16 weeks. ERβ expression was detected in 77% (13 patients). The CB rate at 16 weeks was 15% (2 of 13) in ERβ-positive patients and 0% (0 of 4) in ERβ-negative patients (P = 1). PFS was poor (median, 1.9 months) and not statistically significantly different between ERβ-positive versus -negative patients. No new adverse events from estradiol were identified. The study closed after the first stage as a result of limited responses in these unselected patients. CONCLUSION: In unselected TNBC, high-dose estradiol has limited efficacy. However, further evaluation of ERβ selective agonists in TNBC selected by ERβ expression may be warranted

Targeting the MAPK and PI3K pathways in combination with PD1 blockade in melanoma

Immunotherapy of advanced melanoma with CTLA-4 or PD-1/PD-L1 checkpoint blockade induces in a proportion of patients long durable responses. In contrast, targeting the MAPK-pathway by selective BRAF and MEK inhibitors induces high response rates, but most patients relapse. Combining targeted therapy with immunotherapy is proposed to improve the long-term outcomes of patients. Preclinical data endorsing this hypothesis are accumulating. Inhibition of the PI3K-Akt-mTOR pathway may be a promising treatment option to overcome resistance to MAPK inhibition and for additional combination with immunotherapy. We therefore evaluated to which extent dual targeting of the MAPK and PI3K-Akt-mTOR pathways affects tumor immune infiltrates and whether it synergizes with PD-1 checkpoint blockade in a BRAFV600E/PTEN-/--driven melanoma mouse model. Short-term dual BRAF + MEK inhibition enhanced tumor immune infiltration and improved tumor control when combined with PD-1 blockade in a CD8+ T cell dependent manner. Additional PI3K inhibition did not impair tumor control or immune cell infiltration and functionality. Analysis of on-treatment samples from melanoma patients treated with BRAF or BRAF + MEK inhibitors indicates that inhibitor-mediated T cell infiltration occurred in all patients early after treatment initiation but was less frequent found in on-treatment biopsies beyond day 15. Our findings provide a rationale for clinical testing of short-term BRAF + MEK inhibition in combination with immune checkpoint blockade, currently implemented at our institutes. Additional PI3K inhibition could be an option for BRAF + MEK inhibitor resistant patients that receive targeted therapy in combination with immune checkpoint blockade

Targeting Estrogen Receptor Beta in a Phase 2 Study of High-Dose Estradiol in Metastatic Triple-Negative Breast Cancer: A Wisconsin Oncology Network Study

BACKGROUND: Estrogen receptor beta (ERβ) is expressed by 50-80% of triple negative breast cancers (TNBC). Agonism of ERβ has antiproliferative effects in TNBC cells expressing ERβ. This phase II study evaluated single agent high dose estradiol in patients with advanced TNBC. PATIENTS AND METHODS: Adult women with measurable advanced TNBC were treated with estradiol 10 mg oral three times daily given continuously for 28-day cycles. A Simon optimal two-stage design was used. The primary endpoint was objective response (OR). Secondary endpoints included progression-free survival (PFS), clinical benefit (CB), and safety. OR, CB and PFS by ERβ status were also examined. RESULTS: Seventeen evaluable women were enrolled. Median age was 58 (34-90); the median number of prior systemic therapies was 2 (0-6). One patient had a confirmed partial response (OR rate of 5.9%) and remained on study for >24 weeks. Three patients had stable disease, one lasting more than 16 weeks. ERβ expression was detected in 77% (13 patients). The CB rate at 16 weeks was 15% (2 of 13) in ERβ positive patients and 0% (0 of 4) in ERβ negative patients (p= 1). PFS was poor (median 1.9 months) and not statistically significantly different between ERβ-positive versus negative patients. No new adverse events from estradiol were identified. The study closed after the first stage due to limited responses in these unselected patients. CONCLUSIONS: In unselected TNBC, high dose estradiol has limited efficacy. However, further evaluation of ERβ selective agonists in TNBC selected by ERβ expression may be warranted