INPP5A phosphatase is a synthetic lethal target in GNAQ and GNA11-mutant melanomas.

Activating mutations in GNAQ/GNA11 occur in over 90% of uveal melanomas (UMs), the most lethal melanoma subtype; however, targeting these oncogenes has proven challenging and inhibiting their downstream effectors show limited clinical efficacy. Here, we performed genome-scale CRISPR screens along with computational analyses of cancer dependency and gene expression datasets to identify the inositol-metabolizing phosphatase INPP5A as a selective dependency in GNAQ/11-mutant UM cells in vitro and in vivo. Mutant cells intrinsically produce high levels of the second messenger inositol 1,4,5 trisphosphate (IP3) that accumulate upon suppression of INPP5A, resulting in hyperactivation of IP3-receptor signaling, increased cytosolic calcium and p53-dependent apoptosis. Finally, we show that GNAQ/11-mutant UM cells and patients' tumors exhibit elevated levels of IP4, a biomarker of enhanced IP3 production; these high levels are abolished by GNAQ/11 inhibition and correlate with sensitivity to INPP5A depletion. Our findings uncover INPP5A as a synthetic lethal vulnerability and a potential therapeutic target for GNAQ/11-mutant-driven cancers.

A conditional inducible JAK2V617F transgenic mouse model reveals myeloproliferative disease that is reversible upon switching off transgene expression.

Aberrant activation of the JAK/STAT pathway is thought to be the critical event in the pathogenesis of the chronic myeloproliferative neoplasms, polycythemia vera, essential thrombocythemia and primary myelofibrosis. The most frequent genetic alteration in these pathologies is the activating JAK2V617F mutation, and expression of the mutant gene in mouse models was shown to cause a phenotype resembling the human diseases. Given the body of genetic evidence, it has come as a sobering finding that JAK inhibitor therapy only modestly suppresses the JAK2V617F allele burden, despite showing clear benefits in terms of reducing splenomegaly and constitutional symptoms in patients. To gain a better understanding if JAK2V617F is required for maintenance of myeloproliferative disease once it has evolved, we generated a conditional inducible transgenic JAK2V617F mouse model using the SCL-tTA-2S tet-off system. Our model corroborates that expression of JAK2V617F in hematopoietic stem and progenitor cells recapitulates key hallmarks of human myeloproliferative neoplasms, and exhibits gender differences in disease manifestation. The disease was found to be transplantable, and importantly, reversible when transgenic JAK2V617F expression was switched off. Our results indicate that mutant JAK2V617F-specific inhibitors should result in profound disease modification by disabling the myeloproliferative clone bearing mutant JAK2.

High-Order Drug Combinations Are Required to Effectively Kill Colorectal Cancer Cells.

Like classical chemotherapy regimens used to treat cancer, targeted therapies will also rely upon polypharmacology, but tools are still lacking to predict which combinations of molecularly targeted drugs may be most efficacious. In this study, we used image-based proliferation and apoptosis assays in colorectal cancer cell lines to systematically investigate the efficacy of combinations of two to six drugs that target critical oncogenic pathways. Drug pairs targeting key signaling pathways resulted in synergies across a broad spectrum of genetic backgrounds but often yielded only cytostatic responses. Enhanced cytotoxicity was observed when additional processes including apoptosis and cell cycle were targeted as part of the combination. In some cases, where cell lines were resistant to paired and tripled drugs, increased expression of antiapoptotic proteins was observed, requiring a fourth-order combination to induce cytotoxicity. Our results illustrate how high-order drug combinations are needed to kill drug-resistant cancer cells, and they also show how systematic drug combination screening together with a molecular understanding of drug responses may help define optimal cocktails to overcome aggressive cancers. Cancer Res; 76(23); 6950-63. ©2016 AACR.

CHZ868, a Type II JAK2 Inhibitor, Reverses Type I JAK Inhibitor Persistence and Demonstrates Efficacy in Myeloproliferative Neoplasms.

Although clinically tested JAK inhibitors reduce splenomegaly and systemic symptoms, molecular responses are not observed in most myeloproliferative neoplasm (MPN) patients. We previously demonstrated that MPN cells become persistent to type I JAK inhibitors that bind the active conformation of JAK2. We investigated whether CHZ868, a type II JAK inhibitor, would demonstrate activity in JAK inhibitor persistent cells, murine MPN models, and MPN patient samples. JAK2 and MPL mutant cell lines were sensitive to CHZ868, including type I JAK inhibitor persistent cells. CHZ868 showed significant activity in murine MPN models and induced reductions in mutant allele burden not observed with type I JAK inhibitors. These data demonstrate that type II JAK inhibition is a viable therapeutic approach for MPN patients.

Activity of the Type II JAK2 Inhibitor CHZ868 in B Cell Acute Lymphoblastic Leukemia.

A variety of cancers depend on JAK2 signaling, including the high-risk subset of B cell acute lymphoblastic leukemias (B-ALLs) with CRLF2 rearrangements. Type I JAK2 inhibitors induce paradoxical JAK2 hyperphosphorylation in these leukemias and have limited activity. To improve the efficacy of JAK2 inhibition in B-ALL, we developed the type II inhibitor CHZ868, which stabilizes JAK2 in an inactive conformation. CHZ868 potently suppressed the growth of CRLF2-rearranged human B-ALL cells, abrogated JAK2 signaling, and improved survival in mice with human or murine B-ALL. CHZ868 and dexamethasone synergistically induced apoptosis in JAK2-dependent B-ALLs and further improved in vivo survival compared to CHZ868 alone. These data support the testing of type II JAK2 inhibition in patients with JAK2-dependent leukemias and other disorders.

High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response.

Profiling candidate therapeutics with limited cancer models during preclinical development hinders predictions of clinical efficacy and identifying factors that underlie heterogeneous patient responses for patient-selection strategies. We established ∼1,000 patient-derived tumor xenograft models (PDXs) with a diverse set of driver mutations. With these PDXs, we performed in vivo compound screens using a 1 × 1 × 1 experimental design (PDX clinical trial or PCT) to assess the population responses to 62 treatments across six indications. We demonstrate both the reproducibility and the clinical translatability of this approach by identifying associations between a genotype and drug response, and established mechanisms of resistance. In addition, our results suggest that PCTs may represent a more accurate approach than cell line models for assessing the clinical potential of some therapeutic modalities. We therefore propose that this experimental paradigm could potentially improve preclinical evaluation of treatment modalities and enhance our ability to predict clinical trial responses.

JAK1/2 and Pan-deacetylase inhibitor combination therapy yields improved efficacy in preclinical mouse models of JAK2V617F-driven disease.

PURPOSE: The myeloproliferative neoplasm myelofibrosis is characterized by frequent deregulation of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling, and JAK inhibitors were shown to reduce splenomegaly and ameliorate disease-related symptoms. However, the mutant clone and bone marrow fibrosis persist in the majority of patients. Using preclinical models, we explored whether JAK and pan-deacetylase inhibitor combination yielded additional benefits. EXPERIMENTAL DESIGN: The combination of the JAK1/2 inhibitor ruxolitinib and panobinostat was investigated using two different mouse models of JAK2(V617F)-driven disease. A Ba/F3 JAK2(V617F) cell-driven leukemic disease model was used to identify tolerated and efficacious doses. The drugs were then evaluated alone and in combination in a mouse model of myeloproliferative neoplasm-like disease based on transplantation of bone marrow transduced with a retrovirus expressing JAK2(V617F). Exposures were determined in blood and tissues, and phosphorylated STAT5 and acetylated histone H3 pharmacodynamic readouts were assessed in spleen and bone marrow. Histologic analysis was conducted on spleen and bone marrow, including staining of reticulin fibers in the latter organ. RESULTS: The combination of ruxolitinib and panobinostat was found to have a more profound effect on splenomegaly, as well as on bone marrow and spleen histology, compared with either agent alone, and the analysis of pharmacodynamic readouts showed that ruxolitinib and panobinostat have nonoverlapping and complementary effects. CONCLUSION: Combining JAK1/2 and pan-deacetylase inhibitors was fairly well tolerated and resulted in improved efficacy in mouse models of JAK2(V617F)-driven disease compared with the single agents. Thus, the combination of ruxolitinib and panobinostat may represent a promising novel therapeutic modality for myeloproliferative neoplasms.

In vitro uptake evaluation in Caco-2 cells and in vivo results in diabetic rats of insulin-loaded PLGA nanoparticles.

PLGA nanoparticles (NPs) are largely developed for biological applications but little is known about their uptake. Therefore, we focused our study on the modalities of insulin-loaded PLGA NPs transport across Caco-2 monolayers, and their hypoglycaemic effect on diabetic rats. Insulin-loaded PLGA NPs were formulated by a double emulsion solvent evaporation process. NPs mean diameter was between 130 and 180 nm. NPs were smooth and spherical with an entrapment efficiency above 80%. Fluorescently labeled NPs were incubated with Caco-2 cells to study the process of uptake and the intracellular fate by flow cytometry and confocal laser scanning microscopy. The kinetic of absorption was time-dependent and occurred by clathrin-mediated endocytosis. The intracellular traffic led to a basolateral exocytosis of NPs. In vitro studies and in vivo intraduodenal administration to diabetic rats showed that NPs were resistant in intestinal conditions long enough to allow both the intestinal absorption of NPs and the delivery of functional insulin in bloodstream. The resulting in vivo hypoglycaemic effect was similar to a long-acting insulin one. As no effect on glycaemia occurred after oral administration, further studies need to be conducted to protect NPs from the degradation occurring at the enteric level.

Genetic resistance to JAK2 enzymatic inhibitors is overcome by HSP90 inhibition.

Enzymatic inhibitors of Janus kinase 2 (JAK2) are in clinical development for the treatment of myeloproliferative neoplasms (MPNs), B cell acute lymphoblastic leukemia (B-ALL) with rearrangements of the cytokine receptor subunit cytokine receptor-like factor 2 (CRLF2), and other tumors with constitutive JAK2 signaling. In this study, we identify G935R, Y931C, and E864K mutations within the JAK2 kinase domain that confer resistance across a panel of JAK inhibitors, whether present in cis with JAK2 V617F (observed in MPNs) or JAK2 R683G (observed in B-ALL). G935R, Y931C, and E864K do not reduce the sensitivity of JAK2-dependent cells to inhibitors of heat shock protein 90 (HSP90), which promote the degradation of both wild-type and mutant JAK2. HSP90 inhibitors were 100-1,000-fold more potent against CRLF2-rearranged B-ALL cells, which correlated with JAK2 degradation and more extensive blockade of JAK2/STAT5, MAP kinase, and AKT signaling. In addition, the HSP90 inhibitor AUY922 prolonged survival of mice xenografted with primary human CRLF2-rearranged B-ALL further than an enzymatic JAK2 inhibitor. Thus, HSP90 is a promising therapeutic target in JAK2-driven cancers, including those with genetic resistance to JAK enzymatic inhibitors.