Downregulating Notch counteracts KrasG12D-induced ERK activation and oxidative phosphorylation in myeloproliferative neoplasm.

The Notch signaling pathway contributes to the pathogenesis of a wide spectrum of human cancers, including hematopoietic malignancies. Its functions are highly dependent on the specific cellular context. Gain-of-function NOTCH1 mutations are prevalent in human T-cell leukemia, while loss of Notch signaling is reported in myeloid leukemias. Here, we report a novel oncogenic function of Notch signaling in oncogenic Kras-induced myeloproliferative neoplasm (MPN). We find that downregulation of Notch signaling in hematopoietic cells via DNMAML expression or Pofut1 deletion significantly blocks MPN development in KrasG12D mice in a cell-autonomous manner. Further mechanistic studies indicate that inhibition of Notch signaling upregulates Dusp1, a dual phosphatase that inactivates p-ERK, and downregulates cytokine-evoked ERK activation in KrasG12D cells. Moreover, mitochondrial metabolism is greatly enhanced in KrasG12D cells but significantly reprogrammed by DNMAML close to that in control cells. Consequently, cell proliferation and expanded myeloid compartment in KrasG12D mice are significantly reduced. Consistent with these findings, combined inhibition of the MEK/ERK pathway and mitochondrial oxidative phosphorylation effectively inhibited the growth of human and mouse leukemia cells in vitro. Our study provides a strong rational to target both ERK signaling and aberrant metabolism in oncogenic Ras-driven myeloid leukemia

Targeting BET Proteins Downregulates miR-33a To Promote Synergy with PIM Inhibitors in CMML

Chronic Myelomonocytic Leukemia (CMML) is a rare myeloid malignancy with a dismal prognosis and no therapeutic options which are capable of altering the natural course of the disease. There remains a significant need for novel therapies that are able to meaningfully improve patient outcomes. In this study we explore the effectiveness of Bromodomain and Extra-Terminal domain protein inhibitor (BETi) combinations in CMML. Preclinical studies in myeloid neoplasms have demonstrated efficacy of BETi. However, BETi demonstrate poor single agent activity in clinical trials. Several studies suggest that combinations with other anti-cancer inhibitors may enhance the efficacy of BETi. To nominate BETi combination therapies for myeloid neoplasms, we used a chemical screen with therapies currently in clinical cancer development and validated this screen using a panel of myeloid cell lines, heterotopic cell line models, and PDX models of disease. We identified PIM inhibitors (PIMi) as therapeutically synergistic with BETi in myeloid leukemia models and show that PIM inhibition is able to overcome both single agent BETi and dual BETi/JAKi persistence. Mechanistically, we show that PIM kinase is increased after BETi treatment, and that PIM kinase upregulation is sufficient to induce persistence to BETi and sensitize cells to PIMi. Further, we demonstrate that miR-33a downregulation is the underlying mechanism driving PIM1 upregulation and its downregulation is likely due to global BETi dependent impairments in miRNA biogenesis. We also show that GM-CSF hypersensitivity, a hallmark of CMML, represents a molecular signature for sensitivity to combination therapy. Inhibition of PIM kinases is a potential novel strategy for overcoming BETi persistence in myeloid neoplasms. Our data supports further clinical investigation of this combination

Protein expression of MMP2, TIMP2 in hepatocellular carcinoma: relationships with tumor grade, clinicopathologic parameters, and patient survival.

5009 Background/Aims: Matrix metalloproteinase-2 (MMP-2) is involved in the processes of tumor invasion and metastasis. However, studies on MMP-2 and tissue inhibitor of MMP-2 (TIMP-2) in hepatocellular carcinoma (HCC) are rare, and there are no studies on the correlations with these expressions and HCC patient’s suvival. Methods : We immunohistochemically examined the expression of these proteins and the relationships between these expressions and histological grade, clinicopathological parameters and patient’s survival in 106 patients with HCC. We also examined gelatin zymography and western blotting, using 12 fresh samples of HCC. Results : MMP-2 and TIMP-2 immunoreactivities were presented in HCC cells as well as in non-HCC cells. MMP-2 expression significantly correlated with TIMP-2 expression, and these expressions were stronger in non-tumorous lesions than in tumor lesions. These immunohistochemical results were nearly consistent with the results of western blotting. MMP-2 expression significantly correlated with HCC grade. Though there were no relations between the MMP-2 or TIMP-2 expression and several clinicopathological parameters, overexpressions of MMP-2 and TIMP-2 were associated with poor prognosis of HCC’s patients. Moreovre, TIMP-2 expression was identified as the best predictive factor for determining the prognosis in patients with HCC. Conclusion : MMP-2 and TIMP-2 are involved in HCC progression, and their overexpression is associated with poor prognosis

Phase II study of sunitinib malate (SM) in subjects with metastatic and/or surgically unresectable non-GIST soft tissue sarcomas

10535 Background: Metastatic soft tissue sarcoma represents a diverse collection of disease types. Sunitinib malate is a multitargeted tyrosine kinase inhibitor of vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR) and stem cell factor receptor, with proven efficacy in imatinib mesylate-refractory or intolerant gastrointestinal stromal tumors (GIST). This study was designed to evaluate the efficacy and tolerability of sunitinib malate in four non-GIST sarcoma subytpes (liposarcoma, malignant fibrous histiocytoma, fibrosarcoma and leiomyosarcoma). Methods: Eligibility criteria included tissue documented metastatic soft tissue sarcomas, measurable disease (by RECIST criteria), and a maximum of three prior lines of therapy for metastatic disease. Treatment was with sunitinib malate 50 mg orally daily in 6 week cycles (4 weeks on/ 2 weeks off). Results: Thirty-six subjects have been accrued to date, 12 patients with leiomyosarcoma, 12 with liposarcoma, 11 with..

Rapid Screening of Novel Agents for Combination Therapy in Sarcomas

For patients with sarcoma, metastatic disease remains very difficult to cure, and outcomes remain less than optimal. Treatment options have not largely changed, although some promising gains have been made with single agents in specific subtypes with the use of targeted agents. Here, we developed a system to investigate synergy of combinations of targeted and cytotoxic agents in a panel of sarcoma cell lines. Agents were investigated alone and in combination with varying dose ratios. Dose-response curves were analyzed for synergy using methods derived from Chou and Talalay (1984). A promising combination, dasatinib and triciribine, was explored in a murine model using the A673 cell line, and tumors were evaluated by MRI and histology for therapy effect. We found that histone deacetylase inhibitors were synergistic with etoposide, dasatinib, and Akt inhibitors across cell lines. Sorafenib and topotecan demonstrated a mixed response. Our systematic drug screening method allowed us to screen a large number of combinations of sarcoma agents. This method can be easily modified to accommodate other cell line models, and confirmatory assays, such as animal experiments, can provide excellent preclinical data to inform clinical trials for these rare malignancies

Patient Derived Xenografts (PDX) Recapitulate Ruxolitinib Clinical Trial Responses and Identify a Novel Combination Therapy for Chronic Myelomonocytic Leukemia (CMML)

BACKGROUND: CMML is an aggressive myeloid neoplasm with no known disease modifying treatments. We have shown both that CMML PDXs recapitulate the genetic and pathologic features of this disease, and that the JAK1/2 inhibitor ruxolitinib is a promising therapeutic in a phase 1/2 clinical trial. However, whether CMML PDXs can recapitulate clinical responses is unknown. To explore this, we generated PDXs from bone marrow mononuclear cells (BMMCs) of ruxolitinib clinical trial CMML patients and treated the resultant mice with pharmacologically equivalent doses of ruxolitinib or vehicle. These models were then used to determine if clinical trial responses could be recapitulated and if molecular analysis could identify novel therapeutic strategies for CMML. METHODS: Two million BMMCs from patient samples harvested no later than 30 days before ruxolitinib initiation were transplanted into 3 vehicle and 3 drug-treated NSGS mice as described (Yoshima Blood 2017). Fourteen days post-transplant, mice received either 60 mg/kg ruxolitinib or vehicle via oral gavage twice daily. Treatment continued until the animals became moribund. The primary endpoint of the study was intrapatient PDX overall survival. Secondary PDX response assessments included: (1) reduction in splenomegaly as measured by spleen weight at necropsy, (2) Magnetic Resonance Imaging (MRI) of the spleen at baseline and at the time that the first mouse became moribund (3) and improvement in BM and spleen pathology to include decreases in human CMML engraftment. PDX responses were compared to those seen in the clinical trial per MDS/MPN IWG criteria. Human CD45+ cells from exemplary ruxolitinib- and vehicle-treated mice were sorted and subjected to targeted DNA sequencing and transcriptional and proteomic profiling using the Nanostring Hemeplatform. RESULTS: As we previously reported, the overall response rate of our single-arm ruxolitinib clinical trial (n=50) was 36% and 9 of 23 patients (40%) with splenomegaly had a > 50% reduction in spleen size by physical exam. PDX models were generated from 6 of the patients who had splenomegaly at baseline (3 responders, 3 nonresponders). A total of 34 CMML PDX models were generated and the mean duration on therapy was 63 days (range 7-199 days) for all models. The median overall survival of PDX mice was 59.5 days in the ruxolitinib cohort versus 52 days for the vehicle cohort (HR=1.062, p=0.88). However, ruxolitinib-treated xenografts derived from responders experienced a survival benefit (p=0.0002), a greater reduction in spleen volume when compared to corresponding vehicles (mean decrease in volume -43.9mm3 vs -16.6mm3), and a reduction in bone marrow leukemic engraftment compared to vehicle not observed in PDX mice from nonresponders (p<0.05 vs p=0.90) (Fig. 1 A-C). Metrics assessed at the time of necropsy such as blood counts and spleen weight did not statistically associate by treatment groups. In exemplary mice from 4 patient samples where sufficient cell numbers were recovered at necropsy, DNA-sequencing of sorted human splenocytes confirmed the identical mutations identified in the respective patient sample. In one nonresponding PDX cohort a PI3K/AKT expression signature was identified when comparing ruxolitinib versus vehicle treated mice, suggesting that combining ruxolitinib and PI3K inhibitors could be a viable therapeutic strategy for CMML. To test this, we performed 14-day colony formation assays using 8 unique BMMC CMML patient samples from ruxolitinib nonresponders treated with vehicle, ruxolitinib, and two distinct PI3Kδ inhibitors (PI3Kδi), or their combination. PI3Kδi were chosen as this represented the most highly expressed PI3K isoform in our in-house RNA-seq data set. Interestingly, the combination of ruxolitinib with either PI3Kδi reduced clonogenicity compared to vehicle or each single drug suggesting a novel combination strategy for CMML treatment (Fig. 1D). CONCLUSION: Our CMML PDX platform confirmed the biologic impact of ruxolitinib in CMML, identified a novel combination therapy, and recapitulated clinical responses manifest in human patients. To our knowledge, this is the first report of PDX models recapitulating clinical trial responses in leukemia. A cohort of 6 additional patient samples are currently being assessed to validate these results. Disclosures DeZern: Celgene: Consultancy; Astex Pharmaceuticals, Inc.: Consultancy. Lancet:Daiichi Sankyo: Consultancy, Other: fees for non-CME/CE services ; Pfizer: Consultancy, Research Funding; Agios, Biopath, Biosight, Boehringer Inglheim, Celator, Celgene, Janssen, Jazz Pharmaceuticals, Karyopharm, Novartis: Consultancy. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Roboz:Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amphivena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Actinium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celltrion: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Eisai: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sandoz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Trovagene: Consultancy, Membership on an entity's Board of Directors or advisory committees. Steensma:H3 Biosciences: Other: Research funding to institution, not investigator.; Pfizer: Consultancy; Onconova: Consultancy; Summer Road: Consultancy; Aprea: Research Funding; Stemline: Consultancy; Astex: Consultancy; Arrowhead: Equity Ownership. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Komrokji:JAZZ: Speakers Bureau; DSI: Consultancy; Incyte: Consultancy; pfizer: Consultancy; celgene: Consultancy; Novartis: Speakers Bureau; JAZZ: Consultancy; Agios: Consultancy

Downregulating Notch counteracts KrasG12D-induced ERK activation and oxidative phosphorylation in myeloproliferative neoplasm.

The Notch signaling pathway contributes to the pathogenesis of a wide spectrum of human cancers, including hematopoietic malignancies. Its functions are highly dependent on the specific cellular context. Gain-of-function NOTCH1 mutations are prevalent in human T-cell leukemia, while loss of Notch signaling is reported in myeloid leukemias. Here, we report a novel oncogenic function of Notch signaling in oncogenic Kras-induced myeloproliferative neoplasm (MPN). We find that downregulation of Notch signaling in hematopoietic cells via DNMAML expression or Pofut1 deletion significantly blocks MPN development in KrasG12D mice in a cell-autonomous manner. Further mechanistic studies indicate that inhibition of Notch signaling upregulates Dusp1, a dual phosphatase that inactivates p-ERK, and downregulates cytokine-evoked ERK activation in KrasG12D cells. Moreover, mitochondrial metabolism is greatly enhanced in KrasG12D cells but significantly reprogrammed by DNMAML close to that in control cells. Consequently, cell proliferation and expanded myeloid compartment in KrasG12D mice are significantly reduced. Consistent with these findings, combined inhibition of the MEK/ERK pathway and mitochondrial oxidative phosphorylation effectively inhibited the growth of human and mouse leukemia cells in vitro. Our study provides a strong rational to target both ERK signaling and aberrant metabolism in oncogenic Ras-driven myeloid leukemia

Integrated Human and Murine Clinical Study Establishes Clinical Efficacy of Ruxolitinib in Chronic Myelomonocytic Leukemia

Chronic myelomonocytic leukemia (CMML) is a rare leukemia characterized by peripheral monocytosis with no disease-modifying therapies. CMML cells are uniquely hypersensitive to granulocyte-macrophage colony-stimulating factor (GM-CSF) and robustly engraft in immunocompromised mice that secrete human cytokines. To leverage these unique biological features, we conducted an integrated human and murine study evaluating ruxolitinib, a JAK1/2 inhibitor that potently downregulates intracellular GM-CSF signaling. A total of 50 patients with WHO-defined CMML were enrolled in this open-label, multi-institution phase I/II clinical study, with a ruxolitinib dose of 20 mg twice daily studied in phase II. In parallel, 49 patient-derived xenografts (PDX) derived from 13 study participants were generated and randomized to receive ruxolitinib or vehicle control. The most common grade 3/4 treatment-related toxicities observed were anemia (10%) and thrombocytopenia (6%). The clinical overall response rate was 38% by Myelodysplastic Syndrome/Myeloproliferative Neoplasm (MDS/MPN) International Working Group (IWG) criteria and 43% of patients with baseline splenomegaly achieved a spleen response. Profiling of cytokine levels and somatic mutations at baseline failed to identify predictive biomarkers. PDX models derived from screening samples of study participants recapitulated responses seen in humans, particularly spleen responses, and corroborated ruxolitinib's clinical efficacy in a randomized murine study not feasible in human trials. Ruxolitinib demonstrated clinical efficacy and an acceptable adverse event profile in patients with CMML, identifying a potential novel therapeutic in this rare malignancy. Furthermore, this study demonstrates proof of concept that PDX modeling can recapitulate responses of patients treated on clinical trial and represents a novel correlative study that corroborates clinical efficacy seen in humans.