KIT Activation Research Articles

Emerging Targeted Therapeutic Strategies to Overcome Imatinib Resistance of Gastrointestinal Stromal Tumors.

Gastrointestinal stromal tumors (GISTs) are the most common malignant mesenchymal neoplasms of the gastrointestinal tract. The gold standard for the diagnosis of GISTs is morphologic analysis with an immunohistochemical evaluation plus genomic profiling to assess the mutational status of lesions. The majority of GISTs are driven by gain-of-function mutations in the proto-oncogene c-KIT encoding the tyrosine kinase receptor (TKR) known as KIT and in the platelet-derived growth factor-alpha receptor (PDGFRA) genes. Approved therapeutics are orally available as tyrosine kinase inhibitors (TKIs) targeting KIT and/or PDGFRA oncogenic activation. Among these, imatinib has changed the management of patients with unresectable or metastatic GISTs, improving their survival time and delaying disease progression. Nevertheless, the majority of patients with GISTs experience disease progression after 2-3 years of imatinib therapy due to the development of secondary KIT mutations. Today, based on the identification of new driving oncogenic mutations, targeted therapy and precision medicine are regarded as the new frontiers for GISTs. This article reviews the most important mutations in GISTs and highlights their importance in the current understanding and treatment options of GISTs, with an emphasis on the most recent clinical trials.

Telomere Length and Telomerase Activity of Granulosa Cells and Follicular Fluid in Women Undergoing In Vitro Fertilization

This study aimed to evaluate the interrelationship between telomere length, telomerase activity and oxidative DNA damage in patients undergoing in vitro fertilization (IVF). This single-center, observational clinical study comprised 102 unselected, consecutive patients with various infertility diagnoses. Granulosa cells (GCs) and follicular fluid (FF) were analyzed simultaneously for telomere functions and for the marker of oxidative DNA damage, 8-hydroxy-2-deoxyguanosine (8-OHdG). An Absolute Human Telomere Lengths Quantification qPCR Assay kit and Telomerase Activity Quantification qPCR Assay kit (Nucleotestbio, Budapest, Hungary), as well as an 8-OHdG ELISA kit (Abbexa Ltd., Cambridge, United Kingdom) were used for analyses. Similar telomere lengths were found in GCs and FF, however telomerase activity was markedly depressed, while 8-OHdG levels were markedly elevated in FF compared with those in GCs (p < 0.01). Telomere lengths were independent of telomerase activity both in GCs and FF. However, GC 8-OHdG was inversely related to telomerase activity in GCs and FF (p < 0.05). Importantly, 8-OHdG levels both in GCs and FF had significant negative impact on the number of the retrieved and MII oocytes (p < 0.01), whereas FF 8-OHdG was negatively related further to the number of fertilized oocytes and blastocysts (p < 0.01). In conclusion, we could not confirm the direct association of telomere function and reproductive potential. However, oxidative DNA damage, as mainly reflected by 8-OHdG, adversely affected early markers of IVF outcome and clinical pregnancies.

ETV1 inhibition depressed M2 polarization of tumor-associated macrophage and cell process in gastrointestinal stromal tumor via down-regulating PDE3A.

M2-type polarization of tumor associated-macrophage (TAM) is involved in the malignancy of gastrointestinal stromal tumor (GIST) progression. ETS variant 1 (ETV1) has been previously validated to regulate GIST pathogenesis. Our study intended to explore the role and mechanism of ETV1 in mediating the M2-polarization of TAM in GIST progression. First, we analyzed the correlation between ETV1 expression and M2-polarization in GIST tissues. IL-4 was used to treat THP-1-derived TAM cells and IL-4-stimulated TAM were co-cultured with GIST-T1 cells to mimic the GIST microenvironment. A loss-of-function assay was performed to explore the role of ETV1. Results showed that ETV1 elevation was positively correlated with M2-polarization. IL-4-induced TAM promoted ETV1 expression, silencing ETV1 inhibited proliferation, invasion and KIT activation in IL-4-treated GIST cells, while cell apoptosis was enhanced. Besides, co-culture of ETV1-silenced GIST cells significantly depressed M2-polarization in TAM, presented as decreased levels of CD206, Agr-1 and cytokines, as well as the proportion of CD206-positive TAM. PDE3A was positively correlated with ETV1 and M2-polarization. Overexpressing PDE3A reversed the inhibitory effects of ETV1 silencing. Generally, ETV1 inhibition depressed M2-polarization of TAM in GIST and its promotion on pathological aggravation via down-regulating PDE3A. This evidence may provide a new target for GIST regulation.

Anti-KIT antibody, barzolvolimab, reduces skin mast cells and disease activity in chronic inducible urticaria.

Chronic inducible urticaria (CIndU) is characterized by mast cell (MC)-mediated wheals in response to triggers: cold in cold urticaria (ColdU) and friction in symptomatic dermographism (SD). KIT receptor activation by stem cell factor (SCF) is essential for MC function. Barzolvolimab (CDX-0159) is a humanized antibody that inhibits KIT activation by SCF and was well tolerated in healthy volunteers with dose-dependent plasma tryptase suppression indicative of systemic mast cell ablation. This is an open-label, trial in patients with antihistamine refractory ColdU or SD, receiving one IV dose of barzolvolimab (3 mg/kg), with a 12-week follow-up. Primary endpoint was safety/tolerability; pharmacodynamic (PD)/clinical endpoints included serum tryptase, plasma SCF, skin MC histology, provocation tests, urticaria control test (UCT), and dermatology life quality index (DLQI). Analysis populations were safety (n=21) and pharmacodynamics/clinical activity (n=20). Barzolvolimab was well tolerated; most adverse events were mild and resolved. Treatment resulted in significant depletion of skin MCs, decreased tryptase (<limit of detection), and increased soluble SCF through Week 12. Complete responses (negative provocation test) occurred in 95% (n=19/20) of patients (n=10/10 ColdU; n=9/10 SD), and all (n=20/20) showed improvement in urticaria control (UCT ≥ 12). The kinetics of clinical activity mirrored that of MC and tryptase reduction. DLQI-measured impairment significantly decreased to minimal/none in 93% of patients on study. In CIndU patients, barzolvolimab was well tolerated, demonstrated marked, rapid, durable depletion of skin MCs, circulating tryptase, and reductions in clinical activity with significant improvements in disease control and quality of life (QoL) demonstrating potential therapeutic effects for MC-mediated disorders.

FOXA2 Promotes Prostate Cancer Lineage Plasticity through KIT.

FOXA2 drives the adeno-to-neuroendocrine lineage transition in prostate cancer through KIT activation.

Hyperbaric oxygen protects HT22 cells and PC12 cells from damage caused by oxygen-glucose deprivation/reperfusion via the inhibition of Nrf2/System Xc-/GPX4 axis-mediated ferroptosis.

This study was to investigate the protective effect of hyperbaric oxygen (HBO) on HT22 and PC12 cell damage caused by oxygen-glucose deprivation/reperfusion-induced ferroptosis. A 2-h oxygen-glucose deprivation and 24-h reperfusion model on HT22 and PC12 cells was used to simulate cerebral ischemia-reperfusion injury. Cell viabilities were detected by Cell Counting Kit-8 (CCK-8) method. The levels of reactive oxygen species (ROS) and lipid reactive oxygen species (Lipid ROS) were detected by fluorescent probes Dihydroethidium (DHE) and C11 BODIPY 581/591. Iron Colorimetric Assay Kit, malondialdehyde (MDA) and glutathione (GSH) activity assay kits were used to detect intracellular iron ion, MDA and GSHcontent. Cell ferroptosis-related ultrastructures were visualized using transmission electron microscopy (TEM). Furthermore, PCR and Western blot analyses were used to detect the expressions of ferroptosis-related genes and proteins. After receiving oxygen-glucose deprivation/reperfusion, the viabilities of HT22 and PC12 cells were significantly decreased; ROS, Lipid ROS, iron ions and MDA accumulation occurred in the cells; GSH contents decreased; TEM showed that cells were ruptured and blebbed, mitochondria atrophied and became smaller, mitochondrial ridges were reduced or even disappeared, and apoptotic bodies appeared. And the expressions of Nrf2, SLC7A11 and GPX4 genes were reduced; the expressions of p-Nrf2/Nrf2, xCT and GPX4 proteins were reduced. Notably, these parameters were significantly reversed by HBO, indicating that HBO can protect HT22 cells and PC12 cells from damage caused by oxygen-glucosedeprivation/reperfusion via the inhibition of Nrf2/System Xc-/GPX4 axis-mediated ferroptosis.

The microphthalmia-associated transcription factor is involved in gastrointestinal stromal tumor growth.

Gastrointestinal stromal tumors (GISTs) are the most common neoplasms of mesenchymal origin, and most of them emerge due to the oncogenic activation of KIT or PDGFRA receptors. Despite their relevance in GIST oncogenesis, critical intermediates mediating the KIT/PDGFRA transforming program remain mostly unknown. Previously, we found that the adaptor molecule SH3BP2 was involved in GIST cell survival, likely due to the co-regulation of the expression of KIT and Microphthalmia-associated transcription factor (MITF). Remarkably, MITF reconstitution restored KIT expression levels in SH3BP2 silenced cells and restored cell viability. This study aimed to analyze MITF as a novel driver of KIT transforming program in GIST. Firstly, MITF isoforms were characterized in GIST cell lines and GIST patients' samples. MITF silencing decreases cell viability and increases apoptosis in GIST cell lines irrespective of the type of KIT primary or secondary mutation. Additionally, MITF silencing leads to cell cycle arrest and impaired tumor growth in vivo. Interestingly, MITF silencing also affects ETV1 expression, a linage survival factor in GIST that promotes tumorigenesis and is directly regulated by KIT signaling. Altogether, these results point to MITF as a key target of KIT/PDGFRA oncogenic signaling for GIST survival and tumor growth.

Assessing the Activation of Tyrosine Kinase KIT through Free Energy Calculations.

KIT is a type 3 receptor tyrosine kinase that plays a crucial role in cellular growth and proliferation. Mutations in KIT can dysregulate its active–inactive equilibrium. Activating mutations drive cancer growth, while deactivating mutations result in the loss of skin and hair pigmentation in a disease known as piebaldism. Here, we propose a method based on molecular dynamics and free energy calculations to predict the functional effect of KIT mutations. Our calculations may have important clinical implications by defining the functional significance of previously uncharacterized KIT mutations and guiding targeted therapy.

Abstract A024: In vitro efficacy of a novel dual PARP-HDAC inhibitor in ewing sarcoma

Abstract Introduction: Inhibition of poly-adenosine diphosphate-ribose polymerase (PARP) is an effective therapy against cancers with DNA damage repair (DDR) deficiencies, such as BRCA1 and BRCA2 defects. In preclinical studies, PARP inhibitors demonstrated potential therapeutic value in Ewing sarcoma (ES), though clinical trials with olaparib failed to show significant clinical benefit. While single agent therapy proved inefficacious in the clinical treatment of ES, combination therapies may show anti-tumour activity. A key regulatory event in DNA damage repair is acetylation and deacetylation of histones, controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Increased expression of HDACs have been correlated to more malignant phenotypes in sarcomas and inhibition of HDAC in ES has been shown to be effective in inhibiting tumor growth. HDAC inhibition combined with PARP inhibition has been shown to sensitize cells to treatment in vitro, however clinically, combination therapies often require sequential administration due to different pharmacokinetic profiles and overlapping toxicities, severely limiting clinical utility. Here, we evaluate the activity and efficacy of a novel bifunctional small-molecule compound designed to have both PARP and HDAC inhibiting activity. Methods: PARP1 activity was measured using the Trevigen Universal Colorimetric PARP Assay Kit and PARP2 activity was measured using the BPS Bioscience PARP2 Colorimetric PARP2 Assay Kit. HDAC activity was measured using HeLa nuclear extracts and a fluorogenic peptide-based biochemical assay. Cell survival EC50s were determined using live cell imaging with an Incucyte S3 system and the CellTiter Glo viability assay. Accumulation of phospho-histone H2AX (pH2AX) was detected by western blot using anti-phospho histone H2AX (Ser139) antibody from Cell Signaling Technologies. Results: A representative compound from the kt-3000 series showed potent inhibition of PARP1 and PARP2 with IC50 values in the low nM range, comparable to FDA-approved PARP inhibitors. The compound also showed inhibition of HDAC enzymes with IC50 values in the low µM range, slightly lower than the FDA-approved HDAC inhibitor, vorinostat. Cell survival EC50 values were superior to olaparib in ES cell lines in vitro. Treatment with the kt-3000 compound also resulted in the increased accumulation of pH2AX by western blot and increased S and G2/M cell cycle arrest compared to olaparib. Conclusion: Our kt-3000 compound shows potent inhibition of PARP1, PARP2, and HDAC, as well as induction of DNA damage and cell cycle arrest. Further development of these bifunctional single molecule inhibitors may result in a novel treatment opportunity for Ewing sarcoma. Citation Format: Sarah Truong, Beibei Zhai, Fariba Ghaidi, Louise Ramos, Jay Joshi, Dennis Brown, Neil Sankar, John Langlands, Jeffrey Bacha, Wang Shen, Poul Sorensen, Mads Daugaard. In vitro efficacy of a novel dual PARP-HDAC inhibitor in ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A024.

Abstract A017: Multiple intratumoral sources of kit ligand promote oncogenic kit signaling in gastrointestinal stomal tumor

Abstract Introduction: Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the Kit receptor. While highly effective, tyrosine kinase inhibitors like imatinib are not curative. The natural ligand for the Kit receptor is Kit Ligand (KitL), which exists in both soluble and membrane-bound forms. We sought to determine the role of KitL in the oncogenic Kit signaling of GIST. Methods: KitV558Δ/+ mice, which spontaneously develop an intestinal GIST, were treated with imatinib or vehicle for 1 week, and sorted non-immune (CD45-) cells from tumors were submitted for single-cell RNA sequencing (n=3/group). GIST T1 cells were treated with KitL with or without imatinib and oncogenic Kit signaling was evaluated. KitV558Δ/+ mice were crossed with various KitL mutant and inducible models and tumor weights were measured (n=3-4/group). Human GISTs were stained for KitL expression by IHC, and an existing bulk RNA sequencing human GIST dataset with matched human serum was analyzed. Results: Unsupervised clustering revealed endothelial, smooth muscle, and tumor cells had high expression of KitL RNA in untreated tumors from KitV558Δ/+ mice. Imatinib therapy increased KitL RNA in all 3 cell types, suggesting that extra-tumoral KitL expression is dependent on oncogenic signaling (p&amp;lt;0.05). In GIST T1 cells, exogenous KitL increased Kit activation and reduced imatinib’s efficacy. Tumors from mice with homozygous KitL deletion (KitV558Δ/+;Etv1Cre-ERT2/+;KitLflox/flox) were smaller than tumors from littermates with heterozygous KitL deletion (KitV558Δ /+;Etv1Cre-ERT2/+;KitLflox/+), providing evidence that KitL contributes to GIST development in vivo despite the presence of mutated Kit (p&amp;lt;0.05). Furthermore, tumors from KitV558Δ/+;KitLKitL1Δ/KitL1Δ mice, which lack the proteolytic cleavage site for soluble KitL, were smaller than matched KitV558Δ/+ mice, indicating that soluble KitL is important for maximal tumor growth in vivo (p&amp;lt;0.05). Heterogeneous KitL expression was confirmed in human GISTs by IHC. Human serum contained more KitL in patients with tumors resistant to imatinib and in those with tumors expressing more KitL RNA(p&amp;lt;0.05). Conclusions: KitL is produced by multiple cell types within GIST and contributes to oncogenesis. Targeting KitL may be important for optimal tumor therapy. Citation Format: Andrew D. Tieniber, Ferdinando Rossi, Andrew Hanna, Mengyuan Liu, Mark Etherington, Kevin Do, Laura Wang, Ronald P. DeMatteo. Multiple intratumoral sources of kit ligand promote oncogenic kit signaling in gastrointestinal stomal tumor [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A017.

1489MO A phase Ib/II study of selinexor as single agent and in combination with imatinib in patients with advanced gastrointestinal stromal tumor (GIST): SeliGIST/GEIS-41 trial

Currently approved agents in metastatic GIST focus on targeted suppression of KIT or PDGFRA oncogenic activation. However, their clinical benefit after imatinib (IM) progression is still modest, suggesting the co-operation of KIT/PDGFRA-independent mechanisms in GIST cell survival. Selinexor is an oral, selective inhibitor of XPO1-mediated nuclear export, and preclinical studies evidenced antitumoral activity in GIST as single agent and in combination with IM in both IM-sensitive and IM-resistant models.

Miltirone Attenuates Reactive Oxygen Species-Dependent Neuronal Apoptosis in MPP+-Induced Cell Model of Parkinson's Disease Through Regulating the PI3K/Akt Pathway.

Miltirone is a phenanthrene-quinone derived from Salvia miltiorrhiza Bunge with anti-inflammatory and anti-oxidant effects. Our study aimed to explore the protective effect of miltirone on 1-methyl-4-phenylpyridinium (MPP+)-induced cell model of Parkinson's disease (PD). PharmMapper database was employed to predict the targets of miltirone. PD-related genes were identified using GeneCards database. The overlapping genes between miltirone and PD were screened out using Venn diagram. KEGG analysis was performed using DAVID and KOBAS databases. Cell viability, reactive oxygen species (ROS) generation, apoptosis, and caspase-3 activity were detected by CCK-8 assay, a ROS assay kit, TUNEL, and caspase-3 activity assay, respectively. Effect of miltirone on the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway was explored by western blot analysis. A total of 214 targets of miltirone and 372 targets related to PD were attained, including 29 overlapping targets. KEGG analysis demonstrated that the 29 overlapping targets were both significantly enriched in the PI3K/Akt pathway. MPP+ stimulation reduced the cell viability in SH-SY5Y cells and neuronal primary cultures derived from human brain. Miltirone or N-acetylcysteine (NAC) attenuated MPP+-induced reduction in cell viability, ROS production, SOD activity reduction, apoptosis, and increase of caspase-3 activity. Additionally, miltirone recuperated MPP+-induced inactivation of the PI3K/Akt pathway. Moreover, treatment with LY294002, an inhibitor of the PI3K/Akt pathway, reversed the inhibitory effect of miltirone on MPP+-induced ROS generation and apoptosis in SH-SY5Y cells and neuronal primary cultures. In conclusion, miltirone attenuated ROS-dependent apoptosis in MPP+-induced cellular model of PD through activating the PI3K/Akt pathway.

Long noncoding RNA SNHG14 knockdown exerts a neuroprotective role in MPP+-induced Parkinson's disease cell model through mediating miR-135b-5p/KPNA4 axis.

Parkinson's disease (PD) is a neurodegenerative disease resulted from the loss of dopaminergic neurons. Here, we analyzed the role of long noncoding RNA (lncRNA) small nucleolar RNA host gene 14 (SNHG14) in PD using 1-methyl-4-phenyl pyridine (MPP+)-induced PD cell model. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot assay were performed to determine RNA and protein expression, respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry (FCM) analysis were conducted to analyze cell viability and apoptosis. Enzyme-Linked Immunosorbent Assay (ELISA) was conducted to analyze the release of inflammatory cytokines. Cytotoxicity was assessed using reactive oxygen species (ROS) assay kit, superoxide dismutase (SOD) activity assay kit and lactate dehydrogenase (LDH) activity assay kit. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to confirm the interaction between microRNA-135b-5p (miR-135b-5p) and SNHG14 or karyopherin subunit alpha 4 (KPNA4). MPP+ treatment elevated the expression of SNHG14 in SK-N-SH cells in a dose and time-dependent manner. SNHG14 knockdown alleviated MPP+-induced apoptosis, inflammation, and cytotoxicity in SK-N-SH cells. SNHG14 interacted with miR-135b-5p, and SNHG14 silencing-mediated effects were partly overturned by miR-135b-5p knockdown in PD cell model. Besides, miR-135b-5p interacted with the 3' untranslated region (3'UTR) of KPNA4, and KPNA4 overexpression partly reversed miR-135b-5p overexpression-induced effects in PD cell model. SNHG14 knockdown reduced the protein level of KPNA4 partly by up-regulating miR-135b-5p in SK-N-SH cells. SNHG14 promoted MPP+-induced neuro injury in PD cell model through mediating miR-135b-5p/KPNA4 axis.

The GIST of Advances in Treatment of Advanced Gastrointestinal Stromal Tumor.

Gastrointestinal stromal tumor (GIST) is the most common malignant neoplasm of mesenchymal origin and a compelling clinical and biologic model for the rational development of molecularly targeted agents. This is because the majority of GISTs are driven by gain-of-function mutations in KIT or PDGFRA receptor tyrosine kinases. Specific GIST mutations circumscribe well-defined molecular subgroups that must be determined during the diagnostic work-up to guide clinical management, including therapeutic decisions. Surgery is the cornerstone treatment in localized disease and can also be clinically relevant in the metastatic setting. The correct combination and sequence of targeted agents and surgical procedures improves outcomes for patients with GIST and should be discussed individually within multidisciplinary expert teams. All currently approved agents for the treatment of GIST are based on orally available tyrosine kinase inhibitors targeting KIT and PDGFRA oncogenic activation. Although first-line imatinib achieves remarkable prolonged disease control, the benefit of subsequent lines of treatment is more modest. Novel therapeutic strategies focus on overcoming the heterogeneity of KIT or PDGFRA secondary mutations and providing more potent inhibition of specific challenging mutations. This article reviews the current understanding and treatment of GIST, with an emphasis on recent advances.

Abstract CT107: Correlation of biomarkers and clinical benefit of intratumoral BO112 and pembrolizumab in patients with anti PD1 refractory melanoma

Abstract Background: There is a lack of predictive biomarkers for immunotherapy in melanoma. In terms of IHC, there is no strong rational to support the use of PD-L1 expression. BRAF mutations occur in 40-50% of melanomas and the MAPK pathway may also be activated by NRAS mutations. Patients harboring these mutations face usually a worse prognosis. BO-112 is a double stranded synthetic RNA formulated with polyethyleneimine (PEI) that, by mimicking the effect of a viral infection, mobilizes the immune system. Prior data from a phase I trial (NCT02828098) suggest that, when administered intratumorally, it causes an increase in CD8 infiltration and PD-L1 expression. The role of these and other biomarkers is being explored in the present phase II clinical trial. Study Design: Single arm study with BO-112 plus pembrolizumab (NCT04570332) in patients with advanced melanoma in progression to anti-PD1 therapy. Tumors were genotyped by next generation sequencing, whole exome sequencing and tumor mutation burden. Antitumor and immunological effects of the treatment in the tumor microenvironment were assessed by PDL1 and CD8 immunohistochemistry with a paired biopsy performed after 21 days of treatment. Results: A preliminary analysis has been performed, based on patients evaluable for clinical benefit (defined as response or stable disease&amp;gt;16 weeks). Samples from 35 patients have been analyzed, with 24 patients paired biopsies available. Patients with “cold” tumors (PD-L1 negative and CD8 low) at baseline had a trend to lack of clinical benefit. Only basal PD-L1 in the inflammatory component showed a statistically significant correlation with clinical outcome (4/20 (25%) tumors PDL1 IC negative had benefit versus 10/15 (67%) positive), p=0.0053. Fifteen patients had an increase in PD-L1 and 14 patients had increase in CD8 infiltrate after BO-112 treatment; the lack of increase in PDL1 and CD8 after treatment was also predictive of lack of response (p=0.04). BRAF/NRAS driver mutations correlated with positive outcome. Clinical benefit was observed in 4 of 17 (24%) patients not carrying activating mutations whereas 11 out of 18 (61%) patients with BRAF/NRAS activating mutations had clinical benefit (p=0.02), mainly in cutaneous histology (14% versus 65%, p=0.02). Mucosal melanoma patients (n=3) achieved an ORR 66.7% and DCR 100%. The two mucosal melanoma patients with partial response harbored SETD2 mutations and one of them showed extensive cyclic nucleotide-gated (CNGs), indicative of defects in DNA repair pathways. Regarding acral melanoma patients (n=9), no responses were observed, even in the single case with a NRAS mutation. The only patient achieving clinical benefit, with stable disease&amp;gt;16 weeks; had a unique mutation profile, with TP53 (inactivating) and KIT (activating) mutations. Conclusions: Patients basal mutant BRAF/NRAS could have more probability of benefit from BO-112 and pembrolizumab combination. PD-L1 and/or CD8 increase is an early marker of response. These findings could help to select patients in future clinical trials. Further investigation into predictive biomarkers is warranted. Citation Format: Ruth Roman, Philippe Saiag, Caroline Dutriaux, Luis de la Cruz Merino, Eduardo Castanon, Miguel F. Sanmamed, Caroline Robert, Juan F. Rodríguez-Moreno, Pablo Cerezuela-Fuentes, Ana M. Arance, María González Cao, Henry Montaudié, María Pilar López Criado, Julie Charles, Alfonso Berrocal, María del Carmen Álamo de la Gala, Enrique de Miguel, Elisa Funk-Brentano, Sorilla Prey, Eva Muñoz-Couselo, Delvys Rodríguez Abreu, Juan Martín-Liberal, Helena Escuin-Ordinas, Mark Branum, Sonia Macia, Marisol Quintero, Javier Sánchez López, Marya F. Chaney, Beatriz García-Peláez, Marta Vives-Usano, Miguel Ángel Molina, Stéphane Dalle, Iván Márquez-Rodas. Correlation of biomarkers and clinical benefit of intratumoral BO112 and pembrolizumab in patients with anti PD1 refractory 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 CT107.

Abstract 1058: Evaluation of a novel class of bifunctional DNA alkylating agent and PARP inhibitor

Abstract Introduction: Poly-adenosine diphosphate-ribose polymerase (PARP) inhibition is an effective, FDA-approved treatment against cancers with DNA damage repair (DDR) deficiencies, such as BRCA1 or BRCA2 mutations. PARP is a core component of single-strand break repair, so in tumors with DDR deficiencies, double-strand breaks (DSBs) and single-strand breaks (SSBs) accumulate and lead to cell death. Combining PARP inhibition with other DNA-damaging therapies, such as DNA alkylating agents, has proven to be more effective than PARP inhibition alone, as DNA alkylators can lead to the formation of DSBs. However, combination therapies often require sequential administration due to pharmacokinetic considerations and overlapping toxicities, severely limiting their clinical utility. Here, we evaluate a novel class of PARP inhibitors combined with DNA alkylating functionality, in formulation as a single molecule. Methods: PARP1 activity was determined using the Trevigen Universal Colorimetric PARP Assay Kit and PARP2 activity was determined using the BPS Bioscience PARP2 Colorimetric Assay Kit. Methylator release was evaluated by measuring the appearance of a certain degradation product. Cell cycle analysis was performed using propidium iodide staining and quantified using flow cytometry. pH2AX expression as a measure of DNA double strand breaks (DSB) was quantified by flow cytometry using an Alexa Fluor 647-conjugated anti-H2AX phospho (Ser139) antibody from BioLegend. Detection of pH2AX by western blot and immunofluorescence assays was done using anti-phospho histone H2AX (Ser139) antibody from Cell Signaling Technologies. Results: kt-4000 series compounds are potent inhibitors of both PARP1 and PARP2 with IC50 values in the low nM range, comparable to FDA-approved PARP inhibitors. The compounds release methylator in a similar manner as temozolomide, an FDA-approved DNA alkylating agent. They induce S-phase and G2/M cell cycle arrest in MCF7 cells and lead to accumulation of pH2AX by flow cytometry, western blot, and immunofluorescence assays. Conclusion: Our novel class of PARP inhibitors combined with DNA alkylating function show potent inhibition of both PARP1 and PARP2. These compounds also lead to accumulation of H2AX phosphorylation, a sensitive marker for double-strand breaks, and S-phase and G2/M cell cycle arrest. Development of these bifunctional, single molecule therapies may extend the clinical utility of PARP inhibitors to DDR proficient disease and increase efficacy for DDR deficient disease. Citation Format: Sarah Truong, Beibei Zhai, Fariba Ghaidi, Louise Ramos, Jay Joshi, Dennis Brown, Neil Sankar, John Langlands, Jeffrey Bacha, Wang Shen, Mads Daugaard. Evaluation of a novel class of bifunctional DNA alkylating agent and PARP inhibitor [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 1058.

KETERAMPILAN MAHASISWA PGMI DALAM MELAKSANAKAN PRATIKUM DENGAN MENGGUNAKAN KOMPONEN INSTRUMEN TERPADU (KIT) IPA

Science is the one of the discipline which probes the complex natural phenomena, that the learning should facilitate with the certain tools to aid students to understand its complexity. One of the solution to help Science is integrated component instrument (KIT). The research is done to notice the students’ skill in doing their experiment by using KIT. The sample were 30 students of PGMI. The instrument used in this research observation sheets and questionnaire that had been set based on certain aspects. Overall, the questionnaire result was 72% which categorized as good. The observation reported if generally the student’ skill in KIT activity got 64% or characterized by fair place. At last, the highest scores was obtained in attitude aspect either through questionnaire or observation.

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.

Tyrosine kinase inhibitors for the treatment of indolent systemic mastocytosis: Are we there yet?

Indolent systemic mastocytosis (ISM) is the most prevalent form of systemic mastocytosis. Many patients with ISM suffer from mast cell (MC) mediator-related symptoms. In a small number of patients, hematologic progression is seen in the follow-up. In some patients with ISM, symptoms arising from MC-derived mediators including gastrointestinal symptoms, anaphylaxis, and neuropsychiatric symptoms are kept under control with conventional mediator-targeting drugs or MC-stabilizing agents. However, in a substantial number of patients, the symptoms are refractory to such conventional therapy. For these patients, novel drugs and targeted approaches are considered. One reasonable approach may be to apply tyrosine kinase inhibitors directed against KIT and other key kinase targets expressed in neoplastic MCs in ISM. Because MCs in more than 90% of all patients with typical ISM display the KIT D816V mutant receptor, clinically effective KIT-targeting drugs have to be active against this mutant form of KIT. The 2 such most effective and well-studied agents currently available are midostaurin and avapritinib. Other KIT-targeting drugs, such as imatinib or masitinib, are less effective or even noneffective against KIT D816V and are thus recommended for use only in patients with other KIT mutant forms (noncodon 816 mutations) or with wild-type KIT. In the present article, we review the current state in the treatment of ISM with tyrosine kinase inhibitors, with special emphasis on treatment responses and potential adverse effects. In fact, all of these agents also have unique and common adverse effects, and their use to treat patients with ISM should be balanced against their toxicity and short- and long-term safety.

The Anti-KIT Antibody, CDX-0159, Reduces Mast Cell Numbers and Circulating Tryptase and Improves Disease Control in Patients with Chronic Inducible Urticaria (Cindu)

Mast cells (MCs) are effector cells that mediate chronic urticaria, including CIndU. MC differentiation, proliferation, and survival require activation of KIT receptors by stem cell factor (SCF). CDX-0159 is a humanized monoclonal antibody (mAb) inhibiting SCF-dependent KIT activation. A single 3mg/kg IV dose demonstrated a 95% complete response (negative provocation testing) in CIndU patients and was generally well tolerated as presented previously. Here, we present CDX-0159 effect on urticaria control, pharmacodynamics (PD), and pharmacokinetics (PK).