KIT Signaling Pathways Research Articles

Single nucleus/cell RNA-seq of the chicken hypothalamic-pituitary-ovarian axis offers new insights into the molecular regulatory mechanisms of ovarian development.

The hypothalamic-pituitary-ovarian (HPO) axis represents a central neuroendocrine network essential for reproductive function. Despite its critical role, the intrinsic heterogeneity within the HPO axis across vertebrates and the complex intercellular interactions remain poorly defined. This study provides the first comprehensive, unbiased, cell type-specific molecular profiling of all three components of the HPO axis in adult Lohmann layers and Liangshan Yanying chickens. Within the hypothalamus, pituitary, and ovary, seven, 12, and 13 distinct cell types were identified, respectively. Results indicated that the pituitary adenylate cyclase activating polypeptide (PACAP), follicle-stimulating hormone (FSH), and prolactin (PRL) signaling pathways may modulate the synthesis and secretion of gonadotropin-releasing hormone (GnRH), FSH, and luteinizing hormone (LH) within the hypothalamus and pituitary. In the ovary, interactions between granulosa cells and oocytes involved the KIT, CD99, LIFR, FN1, and ANGPTL signaling pathways, which collectively regulate follicular maturation. The SEMA4 signaling pathway emerged as a critical mediator across all three tissues of the HPO axis. Additionally, gene expression analysis revealed that relaxin 3 (RLN3), gastrin-releasing peptide (GRP), and cocaine- and amphetamine regulated transcripts (CART, also known as CARTPT) may function as novel endocrine hormones, influencing the HPO axis through autocrine, paracrine, and endocrine pathways. Comparative analyses between Lohmann layers and Liangshan Yanying chickens demonstrated higher expression levels of GRP, RLN3, CARTPT, LHCGR, FSHR, and GRPR in the ovaries of Lohmann layers, potentially contributing to their superior reproductive performance. In conclusion, this study provides a detailed molecular characterization of the HPO axis, offering novel insights into the regulatory mechanisms underlying reproductive biology.

Decoding the molecular mechanism of stypoldione against breast cancer through network pharmacology and experimental validation

Breast cancer is the primary factor contributing to female mortality worldwide. The incidence has overtaken lung cancer. It is the most difficult illness due to its heterogeneity and is made up of several subtypes, including Luminal A and B, basal-like, Her-2 overexpressed and TNBC. Amongst different breast carcinoma subtypes, TNBC is the most deadly breast cancer subtype. The hostile nature of TNBC is mainly attributed to its lack of three hormonal receptors and hence lack of targeted therapy. Furthermore, the current diagnostic options like radiotherapy, surgery and chemotherapy render unsuccessful due to recurrence, treatment side effects and drug resistance. The majority of anticancer drugs come from natural sources or is developed from them, making nature a significant source of many medicines. Marine-based constituents such as nucleotides, proteins, peptides, and amides are receiving a lot of interest in the field of cancer treatment due to their bioactive properties. The role of stypoldione in this study as a prospective treatment for breast carcinoma was examined, and we sought to comprehend the molecular means/pathways this chemical employs in breast carcinoma. The most promising possibility for an anti-cancer treatment is stypoldione, a marine chemical produced from the brown alga Stypopodium zonale. We investigated stypoldione's mode of action in breast cancer using the network pharmacology method, and we confirmed our research by using a number of computational tools, including UALCAN, cBioportal, TIMER, docking, and simulation. The findings revealed 92 common targets between the chemical and breast cancer target network. Additionally, we found that stypoldione targets a number of unregulated genes in breast cancer, including: ESR1, HSP90AA1, CXCL8, PTGS2, APP, MDM2, JAK2, KDR, LCK, GRM5, MAPK14, KIT, and several signaling pathways such as FOXO signaling pathway, VEGF pathway, calcium signaling pathway, MAPK/ERK pathway and Neuroactive ligand-receptor interaction. The examined medication demonstrated a strong affinity for the major targets, according to a docking analysis. The best hit compound produced a stable protein–ligand pair, as predicted by molecular dynamics simulations. Our results are supported by the fact that when in-vitro assays were done on melanoma using stypoldione compound it was found that its mechanisms of action involved the PI3K/mTOR/Akt and NF-kB pathways. This study was set out to inspect the possible value of stypoldione as a breast cancer cure and to get a deeper understanding of the molecular mechanisms by which this drug acts on breast cancer.

Uncovering the Heterogeneity of Cardiac Lin-KIT+ Cells: A scRNA-seq Study on the Identification of Subpopulations.

The reparative potential of cardiac Lin-KIT+ (KIT) cells is influenced by their population, but identifying their markers is challenging due to changes in phenotype during in vitro culture. Resolving this issue requires uncovering cell heterogeneity and discovering new subpopulations. Single-cell RNA sequencing (scRNA-seq) can identify KIT cell subpopulations, their markers, and signaling pathways. We used 10× genomic scRNA-seq to analyze cardiac-derived cells from adult mice and found 3 primary KIT cell populations: KIT1, characterized by high-KIT expression (KITHI), represents a population of cardiac endothelial cells; KIT2, which has low-KIT expression (KITLO), expresses transcription factors such as KLF4, MYC, and GATA6, as well as genes involved in the regulation of angiogenic cytokines; KIT3, with moderate KIT expression (KITMOD), expresses the cardiac transcription factor MEF2C and mesenchymal cell markers such as ENG. Cell-cell communication network analysis predicted the presence of the 3 KIT clusters as signal senders and receivers, including VEGF, CXCL, and BMP signaling. Metabolic analysis showed that KIT1 has the low activity of glycolysis and oxidative phosphorylation (OXPHOS), KIT2 has high glycolytic activity, and KIT3 has high OXPHOS and fatty acid degradation activity, indicating distinct metabolic adaptations of the 3 KIT populations. Through the systemic infusion of KIT1 cells in a mouse model of myocardial infarction, we observed their involvement in promoting the formation of new micro-vessels. In addition, in vitro spheroid culture experiments demonstrated the cardiac differentiation capacity of KIT2 cells.

Combination of pimitespib (TAS-116) with sunitinib is an effective therapy for imatinib-resistant gastrointestinal stromal tumors.

Despite the effectiveness of imatinib, most gastrointestinal stromal tumors (GISTs) develop resistance to the treatment, mainly due to the reactivation of KIT tyrosine kinase activity. Sunitinib, which inhibits the phosphorylation of KIT and vascular endothelial growth factor (VEGF) receptor, has been established as second-line therapy for GISTs. The recently-developed heat shock protein 90 (HSP90) inhibitor pimitespib (PIM; TAS-116) demonstrated clinical benefits in some clinical trials; however, the effects were limited. The aim of our study was therefore to clarify the effectiveness and mechanism of the combination of PIM with sunitinib for imatinib-resistant GISTs. We evaluated the efficacy and mechanism of the combination of PIM with sunitinib against imatinib-resistant GIST using imatinib-resistant GIST cell lines and murine xenograft models. In vitro analysis demonstrated that PIM and sunitinib combination therapy strongly inhibited growth and induced apoptosis in imatinib-resistant GIST cell lines by inhibiting KIT signaling and decreasing auto-phosphorylated KIT in the Golgi apparatus. In addition, PIM and sunitinib combination therapy enhanced antitumor responses in the murine xenograft models compared to individual therapies. Further analysis of the xenograft models showed that the combination therapy not only downregulated the KIT signaling pathway but also decreased the tumor microvessel density. Furthermore, we found that PIM suppressed VEGF expression in GIST cells by suppressing protein kinase D2 and hypoxia-inducible factor-1 alpha, which are both HSP90 client proteins. In conclusion, the combination of PIM and sunitinib is effective against imatinib-resistant GIST via the downregulation of KIT signaling and angiogenic signaling pathways.

Influence of Lenvatinib on the Functional Reprogramming of Peripheral Myeloid Cells in the Context of Non-Medullary Thyroid Carcinoma

Lenvatinib is a multitarget tyrosine kinase inhibitor (TKI) approved for the treatment of several types of cancers, including metastatic differentiated thyroid cancer (DTC). The intended targets include VEGFR 1-3, FGFR 1-4, PDGFRα, RET, and KIT signaling pathways, but drug resistance inevitably develops and a complete cure is very rare. Recent data has revealed that most of the TKIs have additional 'off-target' immunological effects, which might contribute to a protective antitumor immune response; however, human cellular data are lacking regarding Lenvatinib-mediated immunomodulation in DTC. Here, we investigated in ex vivo models the impact of Lenvatinib on the function of immune cells in healthy volunteers. We found that monocytes and macrophages were particularly susceptible to Lenvatinib, while neutrophiles and lymphocytes were less affected. In tumor-immune cell co-culture experiments, Lenvatinib exerted a broad inhibitory effect on the proinflammatory response in TC-induced macrophages. Interestingly, Lenvatinib-treated cells had decreased cellular M2 membrane markers, whereas they secreted a significantly higher level of the anti-inflammatory cytokine IL-10 upon LPS stimulation. In addition, prolonged exposure to Lenvatinib impaired macrophages survival and phenotypical differentiation, which was accompanied by remarkable morphological changes and suppressed cellular metabolic activity. These effects were mediated by myeloid cell-intrinsic mechanisms which are independent of Lenvatinib's on-target activity. Finally, using specific inhibitors, we argue that dual effects on p38 MAPK and Syk pathways are likely the underlying mechanism of the off-target immunological effects we observed in this study. Collectively, our data show the immunomodulatory properties of Lenvatinib on human monocytes. These insights could be harnessed for the future design of novel treatment strategies involving a combination of Lenvatinib with other immunotherapeutic agents.

Synergistic Targeting of DNA-PK and KIT Signaling Pathways in KIT Mutant Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common and aggressive form of acute leukemia, with a 5-year survival rate of just 24%. Over a third of all AML patients harbor activating mutations in kinases, such as the receptor tyrosine kinases FLT3 (receptor-type tyrosine-protein kinase FLT3) and KIT (mast/stem cell growth factor receptor kit). FLT3 and KIT mutations are associated with poor clinical outcomes and lower remission rates in response to standard-of-care chemotherapy. We have recently identified that the core kinase of the non-homologous end joining DNA repair pathway, DNA-PK (DNA-dependent protein kinase), is activated downstream of FLT3; and targeting DNA-PK sensitized FLT3-mutant AML cells to standard-of-care therapies. Herein, we investigated DNA-PK as a possible therapeutic vulnerability in KIT mutant AML, using isogenic FDC-P1 mouse myeloid progenitor cell lines transduced with oncogenic mutant KIT (V560G and D816V) or vector control. Targeted quantitative phosphoproteomic profiling identified phosphorylation of DNA-PK in the T2599/T2605/S2608/S2610 cluster in KIT mutant cells, indicative of DNA-PK activation. Accordingly, proliferation assays revealed that KIT mutant FDC-P1 cells were more sensitive to the DNA-PK inhibitors M3814 or NU7441, compared with empty vector controls. DNA-PK inhibition combined with inhibition of KIT signaling using the kinase inhibitors dasatinib or ibrutinib, or the protein phosphatase 2A activators FTY720 or AAL(S), led to synergistic cell death. Global phosphoproteomic analysis of KIT-D816V cells revealed that dasatinib and M3814 single-agent treatments inhibited extracellular signal–regulated kinase and AKT (RAC-alpha serine/threonine-protein kinase)/MTOR (serine/threonine-protein kinase mTOR) activity, with greater inhibition of both pathways when used in combination. Combined dasatinib and M3814 treatment also synergistically inhibited phosphorylation of the transcriptional regulators MYC and MYB. This study provides insight into the oncogenic pathways regulated by DNA-PK beyond its canonical role in DNA repair and demonstrates that DNA-PK is a promising therapeutic target for KIT mutant cancers.

Targeting the translational machinery in gastrointestinal stromal tumors (GIST): a new therapeutic vulnerability

Although KIT-mutant GISTs can be effectively treated with tyrosine kinase inhibitors (TKIs), many patients develop resistance to imatinib mesylate (IM) as well as the FDA-approved later-line agents sunitinib, regorafenib and ripretinib. Resistance mechanisms mainly involve secondary mutations in the KIT receptor tyrosine kinase gene indicating continued dependency on the KIT signaling pathway. The fact that the type of secondary mutation confers either sensitivity or resistance towards TKIs and the notion that secondary mutations exhibit intra- and intertumoral heterogeneity complicates the optimal choice of treatment in the imatinib-resistant setting. Therefore, new strategies that target KIT independently of its underlying mutations are urgently needed. Homoharringtonine (HHT) is a first-in-class inhibitor of protein biosynthesis and is FDA-approved for the treatment of chronic myeloid leukemia (CML) that is resistant to at least two TKIs. HHT has also shown activity in KIT-mutant mastocytosis models, which are intrinsically resistant to imatinib and most other TKIs. We hypothesized that HHT could be effective in GIST through downregulation of KIT expression and subsequent decrease of KIT activation and downstream signaling. Testing several GIST cell line models, HHT led to a significant reduction in nascent protein synthesis and was highly effective in the nanomolar range in IM-sensitive and IM-resistant GIST cell lines. HHT treatment resulted in a rapid and complete abolishment of KIT expression and activation, while KIT mRNA levels were minimally affected. The response to HHT involved induction of apoptosis as well as cell cycle arrest. The antitumor activity of HHT was confirmed in a GIST xenograft model. Taken together, inhibition of protein biosynthesis is a promising strategy to overcome TKI resistance in GIST.

Atractylenolide‑1 alleviates gastroparesis in diabetic rats by activating the stem cell factor/c‑kit signaling pathway.

Diabetic gastroparesis (DGP), also known as delayed gastric emptying, is a common complication of diabetes mellitus. There are numerous clinical symptoms associated with DGP, as well as high treatment costs and markedly reduced patient quality of life. However, the pathogenesis of DGP is not clear, thus effective treatment methods are yet to be established. In the present study, a DGP rat model was established in Sprague-Dawley rats by the intraperitoneal injection of streptozotocin (STZ). DGP model rats were treated with different doses of atractylenolide-1 to detect alterations in gastrointestinal function, including gastroparesis, gastric emptying, gastric motility, gastric peristalsis and gastric blood flow. Compared with the DGP group, atractylenolide-1 treatment significantly reduced glycaemia and the level of glycated hemoglobin, as well as restoring gastrointestinal function. Gastroparesis, gastric emptying, gastric motility, gastric peristalsis and gastric blood flow were significantly impaired in the STZ-induced group compared with the vehicle control group. Moreover, the STZ-induced group displayed downregulated expression levels of the DGP indicator KIT proto-oncogene, receptor tyrosine kinase (c-kit), as investigated by immunohistochemistry, and stem cell factor (SCF) protein, as assessed using ELISA, significantly enhanced rat interstitial cells of Cajal (ICC) apoptosis, and significantly altered levels of oxidative stress-related markers (malondialdehyde and superoxide dismutase) in the serum and gastric tissues compared with the vehicle control group. By contrast, treatment with atractylenolide-1 significantly counteracted the effects of DGP on peristalsis, inhibited apoptosis and suppressed oxidative stress by regulating the expression of heme oxygenase 1 in STZ-induced DGP model rats. Further research indicated that atractylenolide-1 regulated oxidative stress reactions and improved gastric function by activating the SCF/c-kit signaling pathway. Collectively, the results of the present study suggested that atractylenolide-1 promoted ICC survival and preserved the structure of the gastric tissue network in a DGP rat model via the SCF/c-kit signaling pathway, providing novel insights for the treatment of DGP.

Combined Inhibition of AKT and KIT Restores Expression of Programmed Cell Death 4 (PDCD4) in Gastrointestinal Stromal Tumor.

Simple SummaryImatinib mesylate (IM, Gleevec) has remained first-line therapy for most gastrointestinal stromal tumor (GIST) patients since 2002. During this time, three additional inhibitors have been approved for treatment of IM-refractory tumors; however, disease stabilization for these agents is measured in months. Once all approved lines of therapy are ineffective, patients with advanced GIST are left without treatment options. Therefore, preventing or delaying development of acquired resistance to IM could have clinical benefits. Activation of the PI3K/AKT signaling pathway has been associated with resistance to IM in GIST. We evaluated MK-4440, a novel AKT inhibitor, in combination with IM in GIST cells and mouse models. Our studies demonstrate superior activity of MK-4440/IM combination in a panel of GIST cell lines caused by cell cycle arrest and elevated PDCD4 expression leading to increased cell death. Furthermore, dual inhibition of KIT and AKT provided impressive disease stabilization in IM-sensitive GIST growing in mice.The majority of gastrointestinal stromal tumor (GIST) patients develop resistance to the first-line KIT inhibitor, imatinib mesylate (IM), through acquisition of secondary mutations in KIT or bypass signaling pathway activation. In addition to KIT, AKT is a relevant target for inhibition, since the PI3K/AKT pathway is crucial for IM-resistant GIST survival. We evaluated the activity of a novel pan-AKT inhibitor, MK-4440 (formerly ARQ 751), as monotherapy and in combination with IM in GIST cell lines and preclinical models with varying IM sensitivities. Dual inhibition of KIT and AKT demonstrated synergistic effects in IM-sensitive and -resistant GIST cell lines. Proteomic analyses revealed upregulation of the tumor suppressor, PDCD4, in combination treated cells. Enhanced PDCD4 expression correlated to increased cell death. In vivo studies revealed superior efficacy of MK-4440/IM combination in an IM-sensitive preclinical model of GIST compared with either single agent. The combination demonstrated limited efficacy in two IM-resistant models, including a GIST patient-derived xenograft model possessing an exon 9 KIT mutation. These studies provide strong rationale for further use of AKT inhibition in combination with IM in primary GIST; however, alternative agents will need to be tested in combination with AKT inhibition in the resistant setting.

A new monoclonal antibody that blocks dimerisation and inhibits c-kit mutation-driven tumour growth

PurposeImatinib, a small-molecule tyrosine kinase inhibitor, has shown good clinical activity by inhibiting adenosine triphosphate (ATP) binding to the receptor. Unfortunately, majority of patients eventually develop drug resistance, which limits the long-term benefits of the tyrosine kinase inhibitors and poses a significant challenge in the clinical management of GIST. The aim of our study was to explore the feasibility of blocking KIT dimerisation upstream of the phosphorylation in imatinib-resistant GIST.MethodKITMAb was prepared using hybridoma technique. The biological function of KITMAb was examined in KIT-dimer-expressing cells constructed by transfecting with liposomes using enzyme linked immunosorbent assay (ELISA), immunohistochemistry, western blot, MTT, Annexin V/FITC, and flow cytometry assay, respectively.ResultsKIT-dimer was expressed in 293 cells transfected with c-kit mutated-type pcDNA3.1. Treatment of KIT-dimer-expressing cells with the KITMAb significantly decreased the expression of both KIT-dimer and other phosphorylated proteins of KIT downstream signalling pathway. Furthermore, KITMAb slowed down cell growth and reduced the proportion of cells in the proliferative phase (S + G2-M). Finally, we also found that KITMAb treatment accelerated cell apoptosis. These results indicate that KITMAb strongly inhibits KIT receptor dimerisation-mediated signalling pathway and cell growth responses in vitro.ConclusionsWe demonstrate c-kit mutation-driven KIT auto-dimerisation prior to tyrosine kinase phosphorylation as same as the procedure in ligand-dependent signalling pathway and describe a monoclonal antibody, KITMAb, with strong affinity to the dimerisation domain of KIT that blocks the important step in both the KIT signalling pathways. Further, the results suggest that treatment with KITMAb may be potentially therapeutic in imatinib-resistant GIST.

Angiotensin Signaling Is Essential for Stress Erythropoiesis but Results in Retention of Dysfunctional Mitochondria in Erythrocytes That Generate Excessive Reactive Oxygen Species

Sickle cell anemia (SCA), caused by a mutant β-globin gene, results in polymerization of the abnormal hemoglobin S and sickle shaped RBCs that cause vascular occlusions, chronic hemolytic anemia and cumulative organ damage. We have reported sickle nephropathy from elevated levels of angiotensin-II (AT), the bioactive peptide of the renin-angiotensin system (RAS), by signaling through the AT receptor-1 (AT1R), in both mice and humans with SCA, and that occurs in the absence of hypertension or hyper-reninemia [Roy et al, AJH, 2018]. Herein, we investigated the mechanisms underlying the hyperangiotensinemia in SCA. We found that AT levels are elevated due to higher oxidized state of its precursor, angiotensinogen (ATGN), which results from the elevated reactive oxygen species (ROS) in SCA. Oxidized ATGN is more rapidly converted to AT. Hence, the high ROS in SCA increases AT production. Blockade of AT-AT1R signaling in SCA mice, either globally (with captopril that reduces AT production, or losartan that blocks AT1R, or by constitutive genetic knockout of AT1R in SCA mice [SCA AT1R-/-]), or in erythroid cells by an erythroid-specific AT1R knockout (AT1Rf/f EpoR Cre+ termed eCre+) in SCA mice (SCA eCre+) resulted in significant reduction in RBC ROS (Figure 1a). These data show that AT-AT1R signaling in turn generates ROS in sickle erythroid cells, thus creating a positive feedback loop of ↑ROS -->↑RAS -->↑ROS, which causes, and perpetuates the hyperangiotensinemia seen in SCA. The AT-mediated ROS-RAS loop is driven largely by sickle erythropoiesis: SCA eCre+ mice have reduced RBC ROS, hence have reduced AT, and do not develop nephropathy. Surprisingly, while global AT1R deficiency in WT mice (WT AT1R-/-) had no RBC phenotype, SCA AT1R-/- mice developed profound anemia, suggesting AT signaling may be important for the stress erythropoiesis (Stress-E) state, present in SCA. AT is known for its role as a renal erythropoietin (Epo) secretagogue, but its role in Stress-E is unknown. Induction of Stress-E in WT AT1R-/- mice (with phenylhydrazine or daily phlebotomies) resulted in higher anemia in WT AT1R-/- mice than in WT AT1R+/+ mice, suggesting AT1R signaling is important for Stress-E, not baseline erythropoiesis (Base-E). However, WT mice with erythroid-specific deficiency of AT signaling (WT eCre+), when stressed, were able to maintain hemoglobin comparable to their controls (WT eCre-), with an exponential increase in Epo level. Epo levels were high in SCA mice to begin with but were insufficient to compensate with loss of AT signaling in SCA AT1R-/- mice, resulting in development of anemia. RNAseq analysis of sorted Stress-E nucleated erythroid precursors and enucleated erythrocytes in WT AT1R-/- mice showed a remarkable downregulation of the Hedgehog, BMP4, KIT and WNT Stress-E signaling pathways, when compared to WT (AT1R+/+) mice, further confirming that AT signaling is critical, and conceivably upstream to these established Stress-E pathways. However, AT-AT1R signaling in Stress-E states is a double-edged sword: on one hand it was essential to sustain erythropoiesis, while on the other hand it resulted in significant ROS production. This was seen in SCA, and in WT mice where Stress-E was associated with higher RBC ROS (Figure 1a). AT is known to activate NADPH oxidase (NOX), to generate ROS in other cell types. But pharmacological inhibition of NOX signaling, or genetic deficiency of Gp22phox (a common subunit of NOX isoforms) in SCA mice did not lower RBC ROS. However, AT-AT1R signaling in Stress-E increased the mitochondrial mass in erythroid precursors, and also resulted in retention of dysfunctional mitochondria with lower membrane potential in the enucleated erythrocytes, a source of high ROS (Figure 1b-c). Hence, AT signaling in Stress-E inhibited mitophagy, which is required for clearance of mitochondria after enucleation. The transcriptome profile corroborated these findings: With Stress-E, WT AT1R-/- mice had upregulation of genes involved in mitophagy, and genes involved in maintaining mitochondrial integrity/quality and cellular redox homeostasis under oxidative stress. Taken together, our results show that AT signaling plays a critical role in increasing erythroid cell mass during Stress-E, but also downregulates mitophagy, antioxidant genes, and results in increased retention of dysfunctional mitochondria, which are the source of high RBC ROS that mediates end-organ injury. Disclosures Kalfa: Forma Therapeutics, Inc: Research Funding; Agios Pharmaceuticals, Inc: Consultancy, Research Funding. Malik:Aruvant Sciences, Forma Therapeutics, Inc.: Consultancy; Aruvant Sciences, CSL Behring: Patents & Royalties.

Discovery of the Oncogenic Parp1, a Target of bcr-abl and a Potential Therapeutic, in mir-181a/PPFIA1 Signaling Pathway

miR-181a is downregulated in leukemia and affects its progression, drug resistance, and prognosis. However, the exact mechanism of its targets in leukemia, particularly in chronic myelogenous leukemia (CML), has not previously been established. Here, we use a multi-omics approach to demonstrate that protein tyrosine phosphatase, receptor type, f polypeptide, leukocyte common antigen (LAR) interacting protein (liprin), alpha 1 (PPFIA1) is a direct target for miR-181a in CML. Phospho-array assay shows that multiple phosphorylated proteins, particularly KIT signaling molecules, were downregulated in PPFIA1 inhibition. Additionally, PPFIA1 bound PARP1, a common molecule downstream of both PPFIA1 and BCR/ABL, to upregulate KIT protein through activation of nuclear factor kappa B (NF-κB)-P65 expression. Targeted inhibition of PPFIA1 and PARP1 downregulated c-KIT level, inhibited CML cell growth, and prolonged mouse survival. Overall, we report a critical regulatory miR-181a/PPFIA1/PARP1/NF-κB-P65/KIT axis in CML, and our preclinical study supports that targeted PPFIA1 and PARP1 may serve as a potential CML therapy.

PLX9486 shows anti-tumor efficacy in patient-derived, tyrosine kinase inhibitor-resistant KIT-mutant xenograft models of gastrointestinal stromal tumors.

The purpose of the present study was to investigate the in vitro and in vivo activity of PLX9486, a tyrosine kinase inhibitor (TKI) targeting both primary KIT exon 9 and 11 and secondary exon 17 and 18 mutations in gastrointestinal stromal tumors (GISTs). Imatinib, a potent inhibitor of mutated KIT, has revolutionized the clinical management of advanced, metastatic GIST. However, secondary resistance develops mainly through acquired mutations in KIT exons 13/14 or exons 17/18. Second-line sunitinib potently inhibits KIT exon 13/14 mutants but is ineffective against exon 17 mutations. In our study, PLX9486 demonstrated in vitro nanomolar potency in inhibiting the growth and KIT phosphorylation of engineered BaF3 cells transformed with KIT exon 17 mutations (p.D816V) and with the double KIT exon 11/17 mutations (p.V560G/D816V). The in vivo efficacy of PLX9486 was evaluated using two imatinib-resistant GIST patient-derived xenograft (PDX) models. In UZLX-GIST9 (KIT: p.P577del;W557LfsX5;D820G), PLX9486 100mg/kg/day resulted in significant inhibition of proliferation. Pharmacodynamic analysis showed a pronounced reduction in mitogen-activated protein kinase (MAPK) activation and other downstream effects of the KIT signaling pathway but no significant effect on KIT Y703 and Y719 phosphorylation. Similarly, in MRL-GIST1 (KIT: p.W557_K558del;Y823D) PLX9486 treatment led to significant tumor regression and strong inhibition of MAPK activation. Interestingly, the inhibitory effect on MAPK activation was evident even after a single dose of PLX9486. In conclusion, PLX9486 showed anti-tumor efficacy in patient-derived imatinib-resistant GIST xenograft models, mainly through inhibition of KIT signaling. These preclinical efficacy data encourage further testing of PLX9486 in the clinical setting.

Abstract 3840: Plocabulin, a tubulin inhibitor, presents antitumor activity in patient-derived xenograft (PDX) models of gastrointestinal stromal tumor (GIST)

Abstract Introduction: Advanced GIST is commonly treated with tyrosine kinase inhibitors (TKI) [e.g. imatinib (IMA)]. With time the vast majority of patients develops TKI-resistance. GIST is generally believed to be resistant to chemotherapy with cytotoxic agents. The aim of our study was to test plocabulin (PLO; PM060184, PharmaMar), a potent cytotoxic tubulin-dynamics modifier, in two PDX models of GIST, characterized by different sensitivity to IMA. Experimental set-up: NMRI nu/nu mice (n=34) were transplanted bilaterally with human xenografts UZLX-GIST3sens (KIT: exon 11 p.W557_V559delinsF; IMA-sensitive) or -GIST9res (KIT: exon 11+17: p.P577del;W557LfsX5;D820G; IMA-resistant). Xenografted animals were randomly assigned to three treatment groups: control [vehicle, 5ml/kg/QW intravenously (i.v.)], IMA (50mg/kg/BID orally) and PLO (16mg/kg/QW, i.v.). Treatment lasted 22 days and the antitumor activity was assessed by tumor volume measurement, histopathology and KIT signaling pathway by Western blotting. Histological response (HR) was evaluated as previously described by Antonescu et al. 2005. Mann Whitney U test was used for statistical analysis with p <0.05 considered as significant. Results: PLO treatment resulted in a reduction of tumor volume to 59% in GIST3sens and to 70% of the baseline volume in the GIST9res model. Good HR (grade 3, 4) was observed in 70% (GIST3sens) and 50% (GIST9res) of tumors. HR obtained with PLO was mainly characterized by necrosis, while IMA produced mainly myxoid degeneration in the sensitive model. In addition, in GIST3sens PLO decreased the microvessel area and increased apoptosis as assessed by immunohistochemistry, which was not observed in GIST9res. In the latter model PLO showed better activity than IMA in terms of tumor volume reduction (59% vs. 146%, p<0.01) and HR (grade 3, 4 in 50% vs. 0%). KIT signaling was not affected by PLO. The experimental drug was well tolerated throughout the experiment at the dose administered. Conclusions: PLO is the first anti-tubulin agent showing antitumor activity in GIST PDX, both in models sensitive or resistant to IMA. The drug causes cytotoxicity in GIST, mainly through necrosis, without affecting KIT signaling. Due to the different modes of action of PLO and established TKI our work provides a scientific rationale to combine PLO and IMA to overcome resistance to small molecule TKI. Citation Format: Agnieszka Wozniak, Yannick Wang, Jasmien Wellens, Yemarshet K. Gebreyohannes, Maria Jose Guillén, Carlos M. Galmarini, Pablo M. Avilés, Maria Debiec-Rychter, Raf Sciot, Patrick Schöffski. Plocabulin, a tubulin inhibitor, presents antitumor activity in patient-derived xenograft (PDX) models of gastrointestinal stromal tumor (GIST) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3840.

Tranilast prevents renal interstitial fibrosis by blocking mast cell infiltration in a rat model of diabetic kidney disease.

Renal interstitial fibrosis is a final pathway that is observed in various types of kidney diseases, including diabetic kidney disease (DKD). The present study investigated the effect of tranilast on renal interstitial fibrosis and the association between its role and mast cell infiltration in a rat model of DKD. A total of 30 healthy 6‑week‑old male Sprague‑Dawley rats were randomly divided into the following four groups: Normal control group; DKD model group; low‑dose tranilast group (200mg/kg/day); and high‑dose tranilast group (400mg/kg/day). The morphological alterations of tubulointerstitial fibrosis were evaluated by Masson's trichrome staining, while mast cell infiltration into the renal tubular interstitium was measured by toluidine blue staining and complement C3a receptor 1 (C3aR) immunohistochemical staining (IHC). The expression of fibronectin (FN), collagen I (Col‑I), stem cell factor (SCF) and proto‑oncogene c‑kit (c‑kit) was detected by IHC, western blotting and reverse transcription‑quantitative‑polymerase chain reaction. The results demonstrated that tubulointerstitial fibrosis and mast cell infiltration were observed in DKD model rats, and this was improved dose‑dependently in the tranilast treatment groups. The expression of FN, Col‑I, SCF and c‑kit mRNA and protein was upregulated in the tubulointerstitium of DKD model rats compared with the normal control rats, and tranilast inhibited the upregulated expression of these markers. Furthermore, the degree of SCF and c‑kit expression demonstrated a significant positive correlation with C3aR‑positive mast cells and the markers of renal interstitial fibrosis. The results of the present study indicate that mast cell infiltration may promote renal interstitial fibrosis via the SCF/c‑kit signaling pathway. Tranilast may prevent renal interstitial fibrosis through inhibition of mast cell infiltration mediated through the SCF/c-kit signaling pathway.

Kinase profiling of liposarcomas using RNAi and drug screening assays identified druggable targets

BackgroundLiposarcoma, the most common soft tissue tumor, is understudied cancer, and limited progress has been made in the treatment of metastatic disease. The Achilles heel of cancer often is their kinases that are excellent therapeutic targets. However, very limited knowledge exists of therapeutic critical kinase targets in liposarcoma that could be potentially used in disease management.MethodsLarge RNAi and small-molecule tyrosine kinase inhibitor screens were performed against the proliferative capacity of liposarcoma cell lines of different subtypes. Each small molecule inhibitor was either FDA approved or in a clinical trial.ResultsScreening assays identified several previously unrecognized targets including PTK2 and KIT in liposarcoma. We also observed that ponatinib, multi-targeted tyrosine kinase inhibitor, was the most effective drug with anti-growth effects against all cell lines. In vitro assays showed that ponatinib inhibited the clonogenic proliferation of liposarcoma, and this anti-growth effect was associated with apoptosis and cell cycle arrest at the G0/G1 phase as well as a decrease in the KIT signaling pathway. In addition, ponatinib inhibited in vivo growth of liposarcoma in a xenograft model.ConclusionsTwo large-scale kinase screenings identified novel liposarcoma targets and a FDA-approved inhibitor, ponatinib with clear anti-liposarcoma activity highlighting its potential therapy for treatment of this deadly tumor.

What is New in Gastrointestinal Stromal Tumor?

The classification "gastrointestinal stromal tumor" (GIST) became commonplace in the 1990s and since that time various advances have characterized the GIST lineage of origin, tyrosine kinase mutations, and mechanisms of response and resistance to targeted therapies. In addition to tyrosine kinase mutations and their constitutive activation of downstream signaling pathways, GISTs acquire a sequence of chromosomal aberrations. These include deletions of chromosomes 14q, 22q, 1p, and 15q, which harbor putative tumor suppressor genes required for stepwise progression from microscopic, preclinical forms of GIST (microGIST) to clinically relevant tumors with malignant potential. Recent advances extend our understanding of GIST biology beyond that of the oncogenic KIT/PDGFRA tyrosine kinases and beyond mechanisms of KIT/PDGFRA-inhibitor treatment response and resistance. These advances have characterized ETV1 as an essential interstitial cell of Cajal-GIST transcription factor in oncogenic KIT signaling pathways, and have characterized the biologically distinct subgroup of succinate dehydrogenase deficient GIST, which are particularly common in young adults. Also, recent discoveries of MAX and dystrophin genomic inactivation have expanded our understanding of GIST development and progression, showing that MAX inactivation is an early event fostering cell cycle activity, whereas dystrophin inactivation promotes invasion and metastasis.

Abstract 2081: Robust activity of BLU-285, a potent and highly selective inhibitor of mutant KIT and PDGFRα, in patient-derived xenograft (PDX) models of gastrointestinal stromal tumor (GIST)

Abstract Objective: Patients with advanced and treatment-resistant GIST are treated with tyrosine kinase inhibitors (TKI) such as imatinib (IMA), sunitinib (SUN) or regorafenib (REG). Resistance to these TKI is mainly caused by the emergence of on-target secondary KIT mutations in exons 13 and 17. We tested the activity of BLU-285 (Blueprint Medicines), a potent and highly selective inhibitor of mutant KIT and PDGFRα, in three PDX models of primary and refractory GIST that respond differently to the approved TKI used in the treatment of GIST. Methods: NMRI nu/nu mice (n=93) were transplanted bilaterally with human xenografts UZLX-GIST3 (KIT: exon 11 p.W557_V559delinsF; IMA-sensitive), GIST2B (KIT: exon 9 p.A502_Y503dup, IMA dose-dependent sensitive) and GIST9 (KIT: exon 11 p.P577del; W557LfsX5; exon 17 D820G; IMA and SUN-resistant). We performed the following experimental comparisons: BLU-285 (10, 30 and 100 mg/kg/qd) vs. control and IMA (50 mg/kg/bid) [GIST3]; BLU-285 (10, 30 and 60 mg/kg/qd) vs. control and IMA (50 and 100 mg/kg/bid or SUN 40 mg/kg/qd) [GIST2B]; and BLU-285 (10 and 30 mg/kg/qd) vs. control, IMA (50 mg/kg/bid) or REG (30 mg/kg/qd) [GIST9]. Animals were dosed orally for 16 days. Activity was assessed by tumor volume measurement, histopathology and Western blotting of the KIT signaling pathway (WB). Mann Whitney U test was used for statistical analysis. A p value <0.05 was considered significant. Results: In all models, BLU-285 resulted in a dose dependent reduction of tumor volume, significant inhibition of proliferation (p < 0.005 compared to the control in all models, at all doses tested), and inhibition of KIT signaling as assessed by WB. In two models, BLU-285 led to a higher histologic response (graded as described previously by Antonescu et al. 2005), a significant increase of apoptosis (p < 0.005 compared to the control in both models, at all doses tested), and a pronounced decrease in MAPK phosphorylation as compared to the control. The activity of BLU-285 was similar (GIST3) or better (GIST2B and GIST9) than a standard dose of IMA. In the IMA-resistant model (GIST9), the anti-tumor effects of BLU-285 were significantly better those seen with either IMA or REG. BLU-285 was well tolerated at all administered doses. Conclusions: BLU-285, an investigational agent, has significant anti-tumor effects in GIST PDX models characterized by different KIT mutations and variable sensitivity to established TKI therapies. These data support the therapeutic rationale for the ongoing Phase I clinical trial being conducted by Blueprint Medicines (NCT02508532) and provide further evidence that BLU-285 has the potential to be an important treatment option for patients with PDGFRα or KIT-driven GIST. Citation Format: Yemarshet K. Gebreyohannes, Agnieszka Wozniak, Madelina-Elena Zhai, Jasmien Wellens, Jasmien Cornillie, Erica Evans, Alexandra K Gardino, Christoph Lengauer, Maria Debiec-Rychter, Raf Sciot, Patrick Schöffski. Robust activity of BLU-285, a potent and highly selective inhibitor of mutant KIT and PDGFRα, in patient-derived xenograft (PDX) models of gastrointestinal stromal tumor (GIST) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2081. doi:10.1158/1538-7445.AM2017-2081

SLUG transcription factor: a pro-survival and prognostic factor in gastrointestinal stromal tumour

Background:The SLUG transcription factor has been linked with the KIT signalling pathway that is important for gastrointestinal stromal tumour (GIST) tumourigenesis. Its clinical significance in GIST is unknown.Methods:Influence of SLUG expression on cell proliferation and viability were investigated in GIST48 and GIST882 cell lines. The association between tumour SLUG expression in immunohistochemistry and recurrence-free survival (RFS) was studied in two clinical GIST series, one with 187 patients treated with surgery alone, and another one with 313 patients treated with surgery and adjuvant imatinib.Results:SLUG downregulation inhibited cell proliferation, induced cell death in both cell lines, and sensitised GIST882 cells to lower imatinib concentrations. SLUG was expressed in 125 (25.0%) of the 500 clinical GISTs evaluated, and expression was associated with several factors linked with unfavourable prognosis. SLUG expression was associated with unfavourable RFS both when patients were treated with surgery alone (HR=3.40, 95% CI=1.67–6.89, P=0.001) and when treated with surgery plus adjuvant imatinib (HR=1.83, 95% CI=1.29–2.60, P=0.001).Conclusions:GIST patients with high tumour SLUG expression have unfavourable RFS. SLUG may mediate pro-survival signalling in GISTs.

Cabozantinib Is Active against Human Gastrointestinal Stromal Tumor Xenografts Carrying Different KIT Mutations.

In the majority of gastrointestinal stromal tumors (GIST), oncogenic signaling is driven by KIT mutations. Advanced GIST is treated with tyrosine kinase inhibitors (TKI) such as imatinib. Acquired resistance to TKI is mainly caused by secondary KIT mutations, but can also be attributed to a switch of KIT dependency to another receptor tyrosine kinase (RTK). We tested the efficacy of cabozantinib, a novel TKI targeting KIT, MET, AXL, and vascular endothelial growth factor receptors (VEGFR), in patient-derived xenograft (PDX) models of GIST, carrying different KIT mutations. NMRI nu/nu mice (n = 52) were bilaterally transplanted with human GIST: UZLX-GIST4 (KIT exon 11 mutation, imatinib sensitive), UZLX-GIST2 (KIT exon 9, imatinib dose-dependent resistance), or UZLX-GIST9 (KIT exon 11 and 17 mutations, imatinib resistant). Mice were grouped as control (untreated), imatinib (50 mg/kg/bid), and cabozantinib (30 mg/kg/qd) and treated orally for 15 days. Cabozantinib resulted in significant tumor regression in UZLX-GIST4 and -GIST2 and delayed tumor growth in -GIST9. In all three models, cabozantinib inhibited the proliferative activity, which was completely absent in UZLX-GIST4 and significantly reduced in -GIST2 and -GIST9. Increased apoptotic activity was observed only in UZLX-GIST4. Cabozantinib inhibited the KIT signaling pathway in UZLX-GIST4 and -GIST2. In addition, compared with both control and imatinib, cabozantinib significantly reduced microvessel density in all models. In conclusion, cabozantinib showed antitumor activity in GIST PDX models through inhibition of tumor growth, proliferation, and angiogenesis, in both imatinib-sensitive and imatinib-resistant models. Mol Cancer Ther; 15(12); 2845-52. ©2016 AACR.