Constitutive Activation Of MAPK Research Articles

Overexpression of CBK1 or deletion of SSD1 confers fludioxonil resistance in yeast by suppressing Hog1 activation

Group III hybrid histidine kinases (HHK3) are known molecular targets of the widely used fungicidal agent fludioxonil which indirectly converts these kinases to a phosphatase form that causes constitutive activation of Hog1 MAPK. To better understand the fungicidal effect of fludioxonil we have screened S. cerevisiae haploid deletion collection for fludioxonil resistant mutant and identified Ssd1 as a critical factor for this. Deletion of SSD1 not only promoted resistance to fludioxonil but also abrogated Hog1 activation and other cellular damages caused by fludioxonil. Our results showed that fludioxonil perturbed the localization of Cbk1 kinase, an essential protein in yeast, at the bud neck triggering the accumulation of Ssd1 in P-bodies. As a result, localized synthesis of Ssd1 bound mRNA encoding cell wall proteins at the polarized growth site was impaired which created a sustained cell wall stress causing constitutive activation of Hog1. Our data, for the first time, clearly indicated the role of Cbk1 upstream of Hog1 and provided a novel paradigm in the mechanism of action of fludioxonil.

DDDR-04. CA-4948 IN COMBINATION WITH BRAF/MEK INHIBITION IN MELANOMA BRAIN METASTASES

Abstract Melanoma brain metastases (MBM) remain a primary driver of melanoma morbidity and mortality. It is estimated that as many as 60% of all metastatic melanoma patients will develop MBM during the course of their disease. Once found, dual checkpoint inhibitor therapy or BRAF/MEK inhibition in BRAF mutated cancers, are the best options currently available. Even with these treatments, the response to therapy is less than what is seen with disease outside the. In BRAF mutated cancers, which constitute 50% of all patients, initial response to targeted therapy is rapid but resistance to therapy develops 3 months sooner than resistance outside of the brain. Novel strategies to prevent onset of resistance or to work in concert with BRAF/MEK inhibition are needed for this treatment population. The exact mechanism of resistance to targeted therapy in MBM is not known, but mutations in EphA2, PI3K, pAKT, and re-activation of MAPK signaling through secondary mechanisms have all been identified as drivers of resistance. One pathway of MAPK reactivation is through activation of phospho-ERK (eIF2alpha kinase) which also activates the JNK pathway. Our group has previously shown that MBM express high levels of the inflammatory transcription factor IRAK-4, the terminal chaperone of the inflammatory myddosome downstream of TLR and IL1R signally. This increase in IRAK-4 results in constitutive activation of NF-KB, JNK, and MAPK. We have additionally shown the novel oral IRAK-4 inhibitor CA-4948 can reach therapeutically relevant concentrations past the blood brain barrier in both tumor-bearing and naïve mice. We additionally show CA-4948 inhibits MAPK expression in melanoma and suppresses the escape pathways pERK and JNK signaling which can contribute to the prevention of resistance to targeted therapy. This study supports further investigation into the combination potential of CA-4948 with BRAF/MEK inhibitors as triplet therapy to delay intracranial resistance.

Abstract CT197: Phase Ib study of LXH254 + trametinib (TMT) in patients (pts) with NRAS-mutant melanoma

Abstract Background: MAPK pathway dysregulation is a characteristic molecular abnormality of melanoma. NRAS mutations in melanoma lead to constitutive MAPK activation and subsequent MEK and ERK activation. Inhibiting the pathway with a combination of BRAF/CRAF and MEK inhibitors (LXH254 and TMT, respectively) has demonstrated synergistic activity in preclinical models of NRAS-mutant melanoma. Methods: We report data from an open-label, Phase Ib study (NCT02974725), that tested LXH254 + TMT combination in a dose-escalation (ESC) part in pts with KRAS- or BRAF-mutant NSCLC or NRAS-mutant melanoma and in a dose-expansion (EXP) part in NRAS-mutant melanoma pts. In ESC, five dose levels were explored: LXH254 200 mg twice daily (BID) + TMT (1 mg or 0.5 mg) daily (QD); LXH254 400 mg BID + TMT (1 mg or 0.5 mg) QD; LXH254 400 mg BID + TMT 1 mg (QD, 2 wk on/2 wk off). Primary objectives were to evaluate safety and tolerability, and determine the recommended dose for expansion (RDE). Secondary objectives were to characterize pharmacokinetics and pharmacodynamics of LXH254 + TMT, and evaluate antitumor activity. Results: As of Dec 9, 2021, 36 and 30 pts were enrolled in ESC and EXP, respectively; median age was 63.5 and 69 y, respectively. Pts in ESC and EXP received a median of 3 (range: 1-6) and 2 (range: 1-7) prior treatments, respectively, including IO (ESC: 88.9% pts; EXP: 96.7% pts). All pts from ESC and 24 pts from EXP discontinued, mainly due to progressive disease (61.1% and 60%, respectively); 6 pts were ongoing in EXP at data cut-off. In ESC, Grade ≥3 (G≥3) DLTs were reported in 6 pts: dermatitis acneiform (n=2), maculopapular rash (n=2), increased lipase (n=1), and Stevens-Johnson syndrome (n=1). The RDEs were LXH254 200 mg BID + TMT 1 mg QD and LXH254 400 mg BID + TMT 0.5 mg QD. Treatment-related adverse events (TRAEs) were reported in 34 pts (94.4%) in ESC and all pts in EXP. G≥3 TRAEs in ESC and EXP were experienced by 19 (52.8%) and 24 (80%) pts, respectively; the most common being rash or dermatitis acneiform (13.9% each) in ESC and rash (33.3%) in EXP. One fatal TRAE (hypovolemic shock) was reported in the LXH254 400 mg BID + TMT 0.5 mg QD group. The ORR in EXP was 46.7% (7 pts; 95% CI: 21.3-73.4) for LXH254 200 mg BID + TMT 1 mg QD and 13.3% (2 pts; 95% CI: 1.7-40.5) for LXH254 400 mg BID + TMT 0.5 mg QD; overall in EXP, 9 pts (30%) achieved PR and 13 pts (43.3%) reported SD. In EXP the median DOR was 3.8 months (95% CI: 2.6-7.4). Exposures of LXH254 (200 mg or 400 mg BID) + TMT (0.5 mg or 1 mg QD) vs LXH254 single agent were comparable, indicating no apparent LXH254-TMT interactions. Limited paired tumor biopsy data (n=8) showed substantial MAPK pathway inhibition (DUSP6). Conclusions: LXH254 in combination with TMT has shown preliminary antitumor activity in the pretreated, NRAS-mutant melanoma population. A Phase II trial of LXH254 combinations in pts with pretreated BRAF- or NRAS-mutant melanoma is currently ongoing (NCT04417621). Citation Format: Filippo de Braud, Christophe Dooms, Rebecca S. Heist, Celeste Lebbe, Martin Wermke, David Planchard, Dirk Schadendorf, Piotr Rutkowski, Jürgen Wolf, Paolo A. Ascierto, Ignacio Gil-Bazo, Shumei Kato, Maria Wolodarski, Meredith McKean, Eva Muñoz Couselo, Martin Sebastian, Armando Santoro, Vesselina Cooke, Luca Manganelli, Kitty Wan, Anil Gaur, Tanay S. Samant, Jaeyeon Kim, Giordano Caponigro, Xuân-Mai Couillebault, Michele Moschetta, Adil Daud. Phase Ib study of LXH254 + trametinib (TMT) in patients (pts) with NRAS-mutant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT197.

Co-inhibition of SMAD and MAPK signaling enhances 124I uptake in BRAF-mutant thyroid cancers.

Constitutive MAPK activation silences genes required for iodide uptake and thyroid hormone biosynthesis in thyroid follicular cells. Accordingly, most BRAFV600E papillary thyroid cancers (PTC) are refractory to radioiodide (RAI) therapy. MAPK pathway inhibitors rescue thyroid-differentiated properties and RAI responsiveness in mice and patient subsets with BRAFV600E-mutant PTC. TGFB1 also impairs thyroid differentiation and has been proposed to mediate the effects of mutant BRAF. We generated a mouse model of BRAFV600E-PTC with thyroid-specific knockout of the Tgfbr1 gene to investigate the role of TGFB1 on thyroid-differentiated gene expression and RAI uptake in vivo. Despite appropriate loss of Tgfbr1, pSMAD levels remained high, indicating that ligands other than TGFB1 were engaging in this pathway. The activin ligand subunits Inhba and Inhbb were found to be overexpressed in BRAFV600E-mutant thyroid cancers. Treatment with follistatin, a potent inhibitor of activin, or vactosertib, which inhibits both TGFBR1 and the activin type I receptor ALK4, induced a profound inhibition of pSMAD in BRAFV600E-PTCs. Blocking SMAD signaling alone was insufficient to enhance iodide uptake in the setting of constitutive MAPK activation. However, combination treatment with either follistatin or vactosertib and the MEK inhibitor CKI increased 124I uptake compared to CKI alone. In summary, activin family ligands converge to induce pSMAD in Braf-mutant PTCs. Dedifferentiation of BRAFV600E-PTCs cannot be ascribed primarily to activation of SMAD. However, targeting TGFβ/activin-induced pSMAD augmented MAPK inhibitor effects on iodine incorporation into BRAF tumor cells, indicating that these two pathways exert interdependent effects on the differentiation state of thyroid cancer cells.

MEK-ing the Most of It: Strategies to Co-target Gαq and MAPK in Uveal Melanoma.

Most uveal melanomas harbor mutations in Gαq and show constitutive MAPK activation. Although MEK inhibition has some efficacy against uveal melanoma, clinical responses are typically poor. The Gαq inhibitor-MEK inhibitor combination showed prolonged suppression of MAPK signaling in preclinical uveal melanoma models and led to improved therapeutic responses.See related article by Hitchman et al., p. 1476.

Abstract 4177: Preclinical development of ABM-1310, a novel BRAF Inhibitor to treat cancer with brain metastasis

Abstract BRAF is a key molecule activated by RAS in the MAPK pathway that mainly controls cell proliferation. BRAF mutation is detected in about 8% of all cancer population, and more than 50% is BRAFv600e, which causes constitutive MAPK activation. BRAFv600 mutation is found in various cancers such as melanoma, colorectal cancer, glioma, lung cancer, thyroid cancer, pancreatic cancer, ovary cancer etc. ABM-1310 is a novel small molecule BRAF inhibitor that specific inhibits BRAFv600 mutant form with high water solubility, cell permeability, and blood brain barrier (BBB) penetration. Although current target or immune-therapies are reported with some variable effects, melanoma with brain metastasis is still a significant challenge in clinic. With high BBB penetration and high exposure in brain, ABM-1310 has good potential to treat tumors bearing BRAFv600 mutation with brain metastasis. In-vitro data shown similar on-target enzyme activity of ABM-1310 to marketed BRAF inhibitors on BRAFv600e, as well as high anti-proliferation activity in multiple cancer cell lines with BRAF V600E mutation like A375, Colo-829, and HT-29 etc. In vivo pharmacology studies demonstrated that ABM-1310 had good potency of tumor growth inhibition with oral dose as low as 1mg/kg/day. ABM-1310 treatment also improved animal overall survival in several cell-line derived xenograft (CDX) models, including subcutaneous, intracranial and intracardiac melanoma models. Non-clinical pharmacokinetics studies also shown ABM-1310 had a favorable ADME profile both in vitro and in vivo in animals. ABM-1310 demonstrated excellent brain penetration in animals. Single-dose, seven-day repeat dose non-GLP studies and four-week GLP toxicity studies in SD rats and beagle dogs all revealed that ABM-1310 had a good safety profile with NOAEL of 100mg/kg/day in SD rats and 30mg/kg/day in beagle dogs. Based on these supportive preclinical study results, IND of ABM-1310 was submitted in 2019 Q4 to investigate its safety in humans as signal reagent, which will be followed by further clinic development of ABM-1310, or combination with other molecules to treat cancers with BRAFv600 mutation, especially with brain metastasis. Citation Format: Yong Hu, Chen Chen, Charles Huang, Min Xu, Youqin Chen, Xiyuan Chen, Li Zhao. Preclinical development of ABM-1310, a novel BRAF Inhibitor to treat cancer with brain metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4177.

The Senescence-associated Secretory Phenotype Mediates Oncogene-induced Senescence in Pediatric Pilocytic Astrocytoma.

Pilocytic astrocytoma is the most common childhood brain tumor, characterized by constitutive MAPK activation. MAPK signaling induces oncogene-induced senescence (OIS), which may cause unpredictable growth behavior of pilocytic astrocytomas. The senescence-associated secretory phenotype (SASP) has been shown to regulate OIS, but its role in pilocytic astrocytoma remains unknown.Experimental Design: The patient-derived pilocytic astrocytoma cell culture model, DKFZ-BT66, was used to demonstrate presence of the SASP and analyze its impact on OIS in pilocytic astrocytoma. The model allows for doxycycline-inducible switching between proliferation and OIS. Both states were studied using gene expression profiling (GEP), Western blot, ELISA, and cell viability testing. Primary pilocytic astrocytoma tumors were analyzed by GEP and multiplex assay. SASP factors were upregulated in primary human and murine pilocytic astrocytoma and during OIS in DKFZ-BT66 cells. Conditioned medium induced growth arrest of proliferating pilocytic astrocytoma cells. The SASP factors IL1B and IL6 were upregulated in primary pilocytic astrocytoma, and both pathways were regulated during OIS in DKFZ-BT66. Stimulation with rIL1B but not rIL6 reduced growth of DKFZ-BT66 cells and induced the SASP. Anti-inflammatory treatment with dexamethasone induced regrowth of senescent cells and inhibited the SASP. Senescent DKFZ-BT66 cells responded to senolytic BCL2 inhibitors. High IL1B and SASP expression in pilocytic astrocytoma tumors was associated with favorable progression-free survival. We provide evidence for the SASP regulating OIS in pediatric pilocytic astrocytoma, with IL1B as a relevant mediator. SASP expression could enable prediction of progression in patients with pilocytic astrocytoma. Further investigation of the SASP driving the unpredictable growth of pilocytic astrocytomas, and its possible therapeutic application, is warranted.

Discovery of potent, orally bioavailable ERK1/2 inhibitors with isoindolin-1-one structure by structure-based drug design

Constitutive activation of MAPK (RAS/RAF/MEK/ERK) pathway is frequently observed in many tumors and thus has become an interesting therapeutic target for cancer therapy. Despite the successful development of BRAF and MEK inhibitors in clinic treatment, resistance often appears to re-enhance ERK1/2 signaling. Inspired by the central role of the ERK1/2 signaling cascade in cancer, we describe the scaffold-hopping generation of a series of isoindolin-1-one ERK1/2 inhibitors. Our new compounds could inhibit proliferation of KRAS and BRAF mutant cells lines at low nanomolar concentrations. Compound 22a possesses acceptable pharmacokinetic profiles and showed considerable in vivo antitumor efficacy in a HCT-116 xenograft model, providing a promising basis for further optimization towards clinical ERK1/2 inhibitors.

The landscape of genetic alterations in ameloblastomas relates to clinical features

Ameloblastoma is a mostly benign, but locally invasive odontogenic tumor eliciting frequent relapses and significant morbidity. Recently, mutually exclusive mutations in BRAF and SMO were identified causing constitutive activation of MAPK and hedgehog signaling pathways. To explore further such clinically relevant genotype-phenotype correlations, we here comprehensively analyzed a large series of ameloblastomas (98 paraffin block of 76 patients) with respect to genomic alterations, clinical presentation, and histological features collected from the archives of three different pathology centers in France, Germany, and Turkey. In good agreement with previously published data, we observed BRAF mutations almost exclusively in mandibular tumors, SMO mutations predominantly in maxillary tumors, and single mutations in EGFR, KRAS, and NRAS. KRAS, NRAS, PIK3CA, PTEN, CDKN2A, FGFR, and CTNNB1 mutations co-occurred in the background of either BRAF or SMO mutations. Strikingly, multiple mutations were exclusively observed in European patients, in solid ameloblastomas and were associated with a very high risk for recurrence. In contrast, tumors with a single BRAF mutation revealed a lower risk for relapse. We here establish a comprehensive landscape of mutations in the MAPK and hedgehog signaling pathways relating to clinical features of ameloblastoma. Our data suggest that ameloblastomas harboring single BRAF mutations are excellent candidates for neo-adjuvant therapies with combined BRAF/MEK inhibitors and that the risk of recurrence maybe stratified based on the mutational spectrum.

Downregulation of dual-specificity phosphatase 4 enhances cell proliferation and invasiveness in colorectal carcinomas.

It is widely accepted that aberrant activation of the Wnt signaling pathway is responsible for the development of precursor lesions of colorectal cancer (CRC). However, the molecular mechanisms involved in the process of progression from these precursor lesions to invasive lesions of CRC are not fully understood. Recently, we reported that constitutive activation of MAPK accompanied by downregulation of dual‐specificity phosphatase 4 (DUSP4), a MAPK phosphatase, contributes to the progression of precursor lesions in the pancreas. In this study, we found that downregulation of DUSP4 was related to constitutive activation of ERKs in CRC cells. Restoration of DUSP4 resulted in inactivation of ERKs, leading to suppression of both proliferation and invasiveness, as shown by treatment with an MEK inhibitor. Furthermore, immunohistochemistry revealed that DUSP4 expression was upregulated in the superficial region of CRC tissue, whereas it was significantly downregulated in the deep region. In contrast, ERKs in the deep region were markedly hyperactivated compared to those in the superficial region. These results suggest that activation of the MAPK signaling pathway caused by downregulation of DUSP4 is responsible for progression of CRCs and would be a promising therapeutic target.

A Core Regulatory Circuit in Glioblastoma Stem Cells Links MAPK Activation to a Transcriptional Program of Neural Stem Cell Identity

Glioblastoma, the most common primary malignant brain tumor, harbors a small population of tumor initiating cells (glioblastoma stem cells) that have many properties similar to neural stem cells. To investigate common regulatory networks in both neural and glioblastoma stem cells, we subjected both cell types to in-vitro differentiation conditions and measured global gene-expression changes using gene expression microarrays. Analysis of enriched transcription factor DNA-binding sites in the promoters of differentially expressed genes was used to reconstruct regulatory networks involved in differentiation. Computational predictions, which were biochemically validated, show an extensive overlap of regulatory circuitry between cell types including a network centered on the transcription factor KLF4. We further demonstrate that EGR1, a transcription factor previously shown to be downstream of the MAPK pathway, regulates KLF4 expression and that KLF4 in turn transcriptionally activates NOTCH as well as SOX2. These results demonstrate how known genomic alterations in glioma that induce constitutive activation of MAPK are transcriptionally linked to master regulators essential for neural stem cell identify.

Balance between IL-3 and type Iinterferons and their interrelationship with FasL dictates lifespan and effector functions of human basophils.

In contrast to eosinophils and neutrophils, the regulation of the lifespan of human basophils is poorly defined, with the exception of the potent anti-apoptotic effect of IL-3 that also promotes pro-inflammatory effector functions and phenotypic changes. Type I IFNs (IFN-α, IFN-β), which are well known for their anti-viral activities, have the capacity to inhibit allergic inflammation. To elucidate whether type I IFNs have the potential to abrogate the lifespan and/or effector functions of human basophils. We cultured human basophils, and for comparison, eosinophils and neutrophils, with IL-3, interferons, FasL and TRAIL, alone or in combination, and studied cell survival, effector functions and signalling pathways involved. Despite an identical pattern of early signalling in basophils, eosinophils and neutrophils in response to different types of interferons, only basophils displayed enhanced apoptosis after type I IFN treatment. IFN-γ prolonged survival of eosinophils but did not affect the lifespan of basophils. IFN-α-mediated apoptosis required STAT1-STAT2 heterodimers and the contribution of constitutive p38 MAPK activity. Whereas the death ligands FasL and TRAIL-induced apoptosis in basophils per se, IFN-α-mediated apoptosis did neither involve autocrine TRAIL signalling nor did it sensitize basophils to FasL-induced apoptosis. However, IFN-α and FasL displayed an additive effect in killing basophils. Interestingly, IL-3, which protected basophils from IFN-α-, TRAIL- or FasL-mediated apoptosis, did not completely block the additive effect of combined IFN-α and FasL treatment. Moreover, we demonstrate that IFN-α suppressed IL-3-induced release of IL-8 and IL-13. In contrast to IFN-α-mediated apoptosis, these inhibitory effects of IFN-α were not dependent on p38 MAPK signalling. Our study defines the unique and granulocyte-type-specific inhibitory and pro-apoptotic function of type I IFNs and their cooperation with death ligands in human blood basophils, which may be relevant for the anti-allergic properties of type I IFNs.

Ph-like ALL-related novel fusion kinase ATF7IP–PDGFRB exhibits high sensitivity to tyrosine kinase inhibitors in murine cells

ATF7IP-PDGFRB is a novel PDGFRB-related fusion gene identified in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with a signature similar to that of Ph1 ALL, so-called Ph-like ALL. When we introduced ATF7IP-PDGFRB, murine Ba/F3 cells acquired the ability to proliferate in an interleukin (IL)-3-independent manner. On the contrary, the expression of wild-type PDGFRB is not sufficient to acquire the ability for IL-3-independent proliferation in Ba/F3 cells. The introduction of ATF7IP-PDGFRB also induces a typical gene expression profile for Ph1-ALL in Ba/F3 cells. A series of biochemical and cell biological experiments revealed the constitutive activation of ATF7IP-PDGFRB as well as downstream signaling molecules, including AKT and MAPK. Although the phosphoinositide 3-kinase inhibitor led to cell death in both cells into which ATF7IP-PDGFRB had been introduced and IL-3-maintained Mock cells, MEK inhibitor selectively led to cell death into which ATF7IP-PDGFRB had been introduced. The introduction of tyrosine to phenylalanine mutations at binding sites of adaptor molecules important in the MAPK pathway located in the PDGFRB portion abolished ATF7IP-PDGFRB-mediated cell transformation, suggesting that MAPK-mediated signals are critical in ATF7IP-PDGFRB-mediated cell transformation. On treatment with tyrosine kinase inhibitors, ATF7IP-PDGFRB-expressing, but not Mock, Ba/F3 cells underwent rapid apoptosis accompanied by reduced phosphorylation of MAPK. Importantly, the sensitivity of ATF7IP-PDGFRB-expressing Ba/F3 cells to imatinib is significantly higher than that of BCR-ABL1-transformed Ba/F3 cells, as assessed by the IC50. Taken together, ATF7IP-PDGFRB has transforming potential via the constitutive activation of MAPK and participates in the pathogenesis of Ph-like ALL. Our observations suggest the therapeutic importance of tyrosine kinase inhibitors and possibly MEK inhibitor for a subset of BCP-ALL harboring PDGFRB-related fusion kinases.

Tristetraprolin Limits Inflammatory Cytokine Production in Tumor-Associated Macrophages in an mRNA Decay-Independent Manner.

Tristetraprolin (TTP) is an inducible zinc finger AU-rich RNA-binding protein essential for enforcing degradation of mRNAs encoding inflammatory chemokines and cytokines. Most studies on TTP center on the connection between mRNA half-life and inflammatory output, because loss of TTP amplifies inflammation by increasing the stability of AU-rich mRNAs. Here, we focused on how TTP controls cytokine and chemokine production in the nonresolving inflammation of cancer using tissue-specific approaches. In contrast with model in vitro macrophage systems, we found constitutive TTP expression in late-stage tumor-associated macrophages (TAM). However, TTP's effects on AU-rich mRNA stability were negligible and limited by constitutive p38α MAPK activity, which was the main driver of proinflammatory cytokine production in TAMs at the posttranscriptional level. Instead, elimination of TTP caused excessive protein production of inflammatory mediators, suggesting TTP-dependent translational suppression of AU-rich mRNAs. Manipulation of the p38α-TTP axis in macrophages has significant effects on the growth of tumors and therefore represents a means to manipulate inflammation in the tumor microenvironment.

Molecular characterization of disseminated pilocytic astrocytomas.

Pilocytic astrocytomas represent the most common paediatric tumours of the central nervous system. Dissemination through the ventricular system occurs rarely in patients with pilocytic astrocytomas; however, it is more common in infants with diencephalic tumours, and is associated with a poor outcome. Despite histological similarities with classic pilocytic astrocytomas, it is still unclear whether disseminated pilocytic astrocytomas may have specific molecular features. Seventeen disseminated pilocytic astrocytomas were investigated using the molecular inversion probe array and screened for the presence of gene fusions (KIAA1549-BRAF) and mutations (BRAF, RAS and FGFR1). Along with evidence of a constitutive MAPK activation in all cases, the molecular inversion probe array, fluorescence in situ hybridization analysis and mutational study revealed KIAA1549-BRAF fusions in 66% and BRAF(V600E) mutations in 5% of cases. No KRAS, HRAS, NRAS or FGFR1 mutations were found. disseminated pilocytic astrocytomas showed genetic features similar to classic pilocytic astrocytoma, including a similar incidence of KIAA1549-BRAF fusions, BRAF mutations and a stable genetic profile. Given common activation of the MAPK pathway, the use of specific inhibitors can be hypothesized for the treatment of disseminated pilocytic astrocytomas, along with standard chemo- and/or radiotherapy.

Phase II randomized trial of MEK inhibitor pimasertib or placebo combined with gemcitabine in the first-line treatment of metastatic pancreatic cancer.

344 Background: In metastatic pancreatic cancer (mPaCa), KRAS mutations lead to constitutive activation of the MAPK pathway in the vast majority of cases. Pimasertib (Pim) is a selective, non-competitive MEK 1/2 inhibitor with potent antitumor activity in preclinical models with constitutive MAPK activation. Methods: Following a phase I, dose-finding part to the trial, 88 patients (pts) with mPaCa were randomized 1:1 to receive Pim 60 mg BID (A, 44 pts) or placebo (B, 44 pts) in combination with weekly gemcitabine (Gem) 1000 mg/m2(7 of 8 wks in cycle 1, then 3 of 4 wks in subsequent cycles), in a phase II setting (NCT01016483). The primary endpoint was progression-free survival (PFS). Response rate (RR), overall survival (OS) and safety were secondary endpoints. Biomarker analysis was an exploratory endpoint. Results: Pt characteristics were balanced (median age 63.5 yrs, males 56%, stage IV at initial diagnosis 75%) except for PS 0, which was more frequent in arm A (59 vs 41%). Time on treatment was longer in arm B (10.6 vs 8.0 wks). A higher proportion of pts in arm A discontinued treatment during the first 4 wks (31 vs 19%), predominantly due to adverse events (AEs) and PD. Median PFS was 3.7 mo in arm A and 2.8 mo in arm B (HR=0.883, 95% CI: 0.549–1.42; p=0.608). No statistically significant differences were observed between arms for OS (median 7.3 mo in arm A vs 8.3 mo in arm B) and RECIST 1.0 RR (9.1% in both arms). Grade ≥3 thrombocytopenia (20.0 vs 0%), vomiting (15.6 vs 4.8%), fatigue (15.6 vs 7.1%), stomatitis (13.3 vs 0%) and diarrhea (11.1 vs 2.4%) were more common in arm A. Typical allosteric MEK inhibitor-related AEs, such as all grade retinal detachment (24.4%) and creatine phosphokinase elevation (20.0%), were observed almost exclusively in arm A. KRAS mutational status did not influence PFS or OS. Conclusions: The primary study endpoint was not met; secondary endpoints did not suggest clinically meaningful differences between arms, except for selected toxicities observed more frequently with the combination of Gem and Pim. The outcome does not support further development of this combination in the first-line setting in mPaCa. Pim is currently in development in other solid tumors. Clinical trial information: NCT01016483.

LNK (SH2B3) Synergizes with TP53 in Suppressing B-Precursor Acute Lymphoblastic Leukemia through IL-7 Receptor Signaling

B-precursor acute lymphoblastic leukemia (B-ALL) is the leading cause of cancer-related deaths in children and commonly has a poor outcome in adults. Gene profiling and exome sequencing of high-risk ALLs led to the recent identification of the Philadelphia chromosome (Ph)-like ALL subtype. These leukemias have gene expression profiles similar to BCR-ABL1-positive (Ph+) ALL, but lack a BCR-ABL1 rearrangement, and often result in poor outcomes. Many Ph-like ALL-associated mutations identified to date are known or predicted to activate oncogenic cytokine receptor signaling pathways, particularly those associated with JAK. The lymphocyte adaptor protein LNK (also called SH2B3) has emerged as a powerful negative regulator of cytokine-mediated JAK2 signaling in hematopoietic stem cells (HSCs). Loss-of-function LNK mutations and deletions have recently been described in B-ALL, and germline loss of LNK contributes to pediatric ALL development. However, the mechanisms by which LNK alterations impact leukemogenesis remain poorly understood. Here we show that LNK synergizes with TP53 and INK4a in suppressing B-ALL development in mice. Tp53-/-Lnk-/- mice developed highly aggressive and transplantable B-ALL with 100% penetrance in contrast to T-lymphoma or sarcoma development observed in Tp53-/- mice. Importantly, gene expression profiles of Tp53-/-Lnk-/- B-ALL blasts were similar to those of human Ph-like B-ALLs, validating the relevance of this model for preclinical studies. Tp53-/-Lnk-/- pro-B progenitors initiated B-ALL in the transplanted recipients, and Lnk loss-of-heterozygosity (LOH) was found exclusively in B-ALL cells from Tp53-/-Lnk+/- mice, but not in HSCs, common lymphoid progenitors, or cells of myeloid/T cell lineages, attesting that LNK is a bona fide tumor suppressor in the committed B-cell precursors. Mechanistically, we found that pre-leukemic Lnk-/-Tp53-/- pro-B progenitors were hypersensitive to IL-7 and showed markedly enhanced self-renewal ability in vitro and in vivo. Our genetic studies revealed that LNK controls normal B cell and B-ALL development independently of its effects on HSCs and specifically regulates pro-B cell homeostasis. A novel phosphoflow cytometry approach in freshly isolated BM cells that combines the surface marker B220, intracellular mu-heavy chain detection, and phospho-specific antibodies enabled separation of subpopulations of B progenitors with distinct IL-7 responsiveness. Using this approach, we demonstrated that LNK deficiency potentiated STAT5 activation in response to IL-7 in pre-leukemic pro-B cells. Of note, Lnk-/-p53-/- leukemic blasts with elevated pSTAT5, were less sensitive to JAK inhibitors than pre-leukemic B progenitors, in part due to constitutive activation of MAPK and AKT/mTOR pathways. Our results invoke the targeting of these pathways as novel therapeutic approaches in B-ALL. In sum, we have developed a novel B-ALL mouse model suitable for preclinical studies aimed at further deciphering the pathogenic mechanisms underlying this disease and exploring new therapeutic strategies. DisclosuresNo relevant conflicts of interest to declare.

A MicroRNA Processing Defect in Smokers' Macrophages Is Linked to SUMOylation of the Endonuclease DICER

Despite the fact that alveolar macrophages play an important role in smoking-related disease, little is known about what regulates their pathophysiologic phenotype. Evaluating smoker macrophages, we found significant down-regulation of multiple microRNAs (miRNAs). This work investigates the hypothesis that cigarette smoke alters mature miRNA expression in lung macrophages by inhibiting processing of primary miRNA transcripts. Studies on smoker alveolar macrophages showed a defect in miRNA maturation. Studies on the miRNA biogenesis machinery led us to focus on the cytosolic RNA endonuclease, DICER. DICER cleaves the stem-loop structure from pre-miRNAs, allowing them to dissociate into their mature 20-22-nucleotide single-stranded form. DICER activity assays confirmed impaired DICER activity following cigarette smoke exposure. Further protein studies demonstrated a decreased expression of the native 217-kDa form of DICER and an accumulation of high molecular weight forms with cigarette smoke exposure. This molecular mass shift was shown to contain SUMO moieties and could be blocked by silencing RNA directed at the primary SUMOylating ligase, Ubc9. In determining the cigarette smoke components responsible for changes in DICER, we found that N-acetylcysteine, an antioxidant and anti-aldehyde, protected DICER protein and activity from cigarette smoke extract. This massive down-regulation of miRNAs (driven in part by alterations in DICER) may be an important regulator of the disease-promoting macrophage phenotype found in the lungs of smokers.

Abstract CN07-04: Maximizing the effectiveness of radioiodine therapy for metastatic thyroid cancer with selective inhibitors of MAPK signaling.

Abstract Thyroid cancers are associated with mutually exclusive point mutations of BRAF (57%) and RAS (NRAS >HRAS; 12%), and fusion events activating BRAF, or the RTKs RET, NTRK1 or NTRK3. The molecular taxonomy of the disease is different in more advanced forms of thyroid cancer. Although BRAF mutations also predominate, poorly differentiated (PDTC) and anaplastic thyroid cancers (ATC) are relatively enriched for RAS mutations (23-44%). As predicted from the genetics of the disease, thyroid cancers, like melanomas, are ERK-driven tumors. However, they exhibit lineage-specific properties that offer distinct therapeutic opportunities and challenges. The 10-year survival of patients with metastatic thyroid cancers that retain radioiodine (RAI) avidity is ∼60%, whereas it is only 10% if the metastases are refractory to RAI therapy (1). There are several lines of evidence indicating that constitutive MAPK activity causes decreased radioiodine responsiveness. Conditional expression of oncogenic RET/PTC, RAS or BRAF in thyroid PCCL3 cells downregulates expression of the sodium iodide symporter (NIS) and other key genes required to organify and retain iodide (2, 3), which is restored by treatment with RAF or MEK inhibitors (2, 4). Mice with dox-inducible expression of BRAFV600E in thyroid cells develop invasive papillary thyroid cancers with profoundly impaired expression of genes required for iodine uptake and metabolism and of 124-iodine uptake in vivo, which are reversed when dox is discontinued, but only partially restored by RAF or MEK inhibitors (5). Based on these findings we performed a phase 2 trial of the MEK inhibitor selumetinib (AZD6244) in patients with RAI-refractory thyroid cancer. Selumetinib increased iodide incorporation at metastatic sites in 12/20 patients as determined by 124I PET scanning, and in ∼40% (8/20) the increase in uptake was sufficient to enable treatment with 131I, with remarkable clinical responses (6). Responses were greater in patients whose tumors harbored RAS as compared to BRAF mutations. An international multicenter phase 3 registration study of selumetinib vs placebo to increase remission rates of high risk thyroid cancer patients receiving post-surgical adjuvant radioiodine therapy is now in progress (the ASTRA trial). There is concern that responses to monotherapy with MEK or RAF inhibitors may not be optimal, particularly in BRAF-mutant disease. We find that BRAF-mutant human thyroid cancer cell lines have decreased responsiveness to RAF and MEK inhibitors because of relief of negative feedback resulting in derepression of HER3 transcription, followed by neuregulin-1- induced HER3/HER2 signaling and reactivation of the MEK-ERK and PI3K-AKT pathways, which is overcome by co-treatment with HER kinase inhibitors (7). This mechanism of adaptive resistance differs in significant ways from the responses seen in BRAF-mutant melanoma and colorectal cell lines. Our preclinical data predicts that optimal restoration of RAI responsiveness would be best achieved by compounds that inhibit MAPK output in a profound and sustained way. Indeed, recent experiments show that such strategies have excellent potential to further augment the specialized functions of thyroid cells, and lead to even more effective therapies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):CN07-04. Citation Format: James A. Fagin. Maximizing the effectiveness of radioiodine therapy for metastatic thyroid cancer with selective inhibitors of MAPK signaling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr CN07-04.

Improvement of Cancer Immunotherapy by Combining Molecular Targeted Therapy

In human cancer cells, a constitutive activation of MAPK, STAT3, β-catenin, and various other signaling pathways triggers multiple immunosuppressive cascades. These cascades result in the production of immunosuppressive molecules (e.g., TGF-β, IL-10, IL-6, VEGF, and CCL2) and induction of immunosuppressive immune cells (e.g., regulatory T cells, tolerogenic dendritic cells, and myeloid-derived suppressor cells). Consequently, immunosuppressive conditions are formed in tumor-associated microenvironments, including the tumor and sentinel lymph nodes. Some of these cancer-derived cytokines and chemokines impair immune cells and render them immunosuppressive via the activation of signaling molecules, such as STAT3, in the immune cells. Thus, administration of signal inhibitors may inhibit the multiple immunosuppressive cascades by acting simultaneously on both cancer and immune cells at the key regulatory points in the cancer-immune network. Since common signaling pathways are involved in manifestation of several hallmarks of cancer, including cancer cell proliferation/survival, invasion/metastasis, and immunosuppression, targeting these shared signaling pathways in combination with immunotherapy may be a promising strategy for cancer treatment.