ASP210: a potent oligonucleotide-based inhibitor effective against TKI-resistant CML cells.

Clinical experience with tyrosine kinase inhibitors (TKIs) over the past two decades has shown that, despite the apparent therapeutic benefit, nearly 30% of patients with chronic myelogenous leukemia (CML) display primary resistance or intolerance to TKIs, and approximately 25% of those treated are forced to switch TKIs at least once during therapy due to acquired resistance. Safe and effective treatment modalities targeting leukemic clones that escape TKI therapy could hence be game changers in the professional management of these patients. Here, we aimed to investigate the efficacy of a novel therapeutic oligonucleotide of unconventional design, called ASP210, to reduce BCR-ABL1 mRNA levels in TKI-resistant CML cells, with the assumption of inducing their apoptosis. Imatinib- and dasatinib-resistant sublines of BCR-ABL1-positive MOLM-7 and CML-T1 cells were established and exposed to 0.25 and 2.5 µM ASP210 for 10 days. RT-qPCR showed a remarkable reduction of the target mRNA level by >99% after a single application. Cell viability was monitored daily by trypan blue staining. In response to the lack of driver oncoprotein BCR-ABL1, TKI-resistant CML cells underwent apoptosis regardless of the presence of the clinically relevant T315I mutation by day 5 after redosing with ASP210. The effect was selective for cancer cells, indicating a favorable safety profile for this therapeutic modality. Furthermore, the spontaneous uptake and high intracellular concentrations of ASP210 suggest its potential to be effective at relatively low doses. The present findings suggest that ASP210 is a promising therapeutic avenue for patients with CML who fail to respond to TKI therapy.NEW & NOTEWORTHY Effective treatment modalities targeting leukemic clones that escape tyrosine kinase inhibitor (TKI) therapy could be game changers in the professional management of patients displaying primary resistance, intolerance, or acquired resistance to TKIs. Although delivering authentic innovations today is more complex than ever, we developed a highly potent and safe oligonucleotide-based modality against BCR-ABL1 mRNA named ASP210 that effectively induces cell death in BCR-ABL1-positive TKI-resistant cells while sparing BCR-ABL1-negative healthy cells.

Investigating the preferential interaction between imatinib mesylate and VEGF G-quadruplex DNA as therapeutic strategies for cancer treatment: Biophysical and molecular modelling approaches.

G-Quadruplex DNA (GQ-DNA) is one of the most important non-canonical nucleic acid structures. GQ-DNA forming sequences are present in different crucial genomic regions and are abundant in promoter regions of several oncogenes. Therefore, GQ-DNA is an important target for anticancer drugs and hence binding interactions between GQ-DNA and small molecule ligands are of great importance. Since GQ-DNA is a highly polymorphic structure, it is important to identify ligand molecules which preferentially target a particular quadruplex sequence. In this present study, we have used a FDA approved drug called imatinib mesylate (ligand) which is a selective tyrosine kinase inhibitor, successfully used for the treatment of chronic myelogenous leukaemia, gastrointestinal stromal tumours. Different spectroscopic techniques as well as molecular docking investigations and molecular simulations have been used to explore the interaction between imatinib mesylate with VEGF GQ DNA structures along with duplex DNA, C-Myc, H-Telo GQ DNA. We found that imatinib mesylate shows preferential interaction towards VEGF GQ DNA compared to C-Myc, H-Telo GQ and duplex DNA. Imatinib mesylate seems to be an efficient ligand for VEGF GQ DNA, suggesting that it might be used to regulate the expression of genes in cancerous cells.

Nutrient-sensitizing drug repurposing screen identifies lomerizine as a mitochondrial metabolism inhibitor of chronic myeloid leukemia.

In chronic myeloid leukemia (CML), the persistence of leukemic stem cells (LSCs) after treatment with tyrosine kinase inhibitors (TKIs), such as imatinib, can lead to disease relapse. It is known that therapy-resistant LSCs rely on oxidative phosphorylation (OXPHOS) for their survival and that targeting mitochondrial respiration sensitizes CML LSCs to imatinib treatment. However, current OXPHOS inhibitors have demonstrated limited efficacy or have shown adverse effects in clinical trials, highlighting that identification of clinically safe oxidative pathway inhibitors is warranted. We performed a high-throughput drug repurposing screen designed to identify mitochondrial metabolism inhibitors in myeloid leukemia cells. This identified lomerizine, a US Food and Drug Administration (FDA)-approved voltage-gated Ca2+ channel blocker now used for the treatment of migraines, as one of the top hits. Transcriptome analysis revealed increased expression of voltage-gated CACNA1D and receptor-activated TRPC6 Ca2+ channels in CML LSCs (CD34+CD38-) compared with normal counterparts. This correlated with increased endoplasmic reticulum (ER) mass and increased ER and mitochondrial Ca2+ content in CML stem/progenitor cells. We demonstrate that lomerizine-mediated inhibition of Ca2+ uptake leads to ER and mitochondrial Ca2+ depletion, with similar effects seen after CACNA1D and TRPC6 knockdown. Through stable isotope-assisted metabolomics and functional assays, we observe that lomerizine treatment inhibits mitochondrial isocitrate dehydrogenase activity and mitochondrial oxidative metabolism and selectively sensitizes CML LSCs to imatinib treatment. In addition, combination treatment with imatinib and lomerizine reduced CML tumor burden, targeted CML LSCs, and extended survival in xenotransplantation model of human CML, suggesting this as a potential therapeutic strategy to prevent disease relapse in patients.

Repurposing approved protein kinase inhibitors as potent anti-leishmanials targeting Leishmania MAP kinases.

AIMS: Leishmaniasis, caused by the protozoan parasite poses a significant health burden globally. With a very few specific drugs, increased drug resistance it is important to look for drug repurposing along with the identification of pre-clinical candidates against visceral leishmaniasis. This study aims to identify potential drug candidates against visceral leishmaniasis by targeting leishmanial MAP kinases and screening FDA approved protein kinase inhibitors. MATERIALS AND METHODS: MAP kinases were identified from the Leishmania genome. 12 FDA approved protein kinase inhibitors were screened against Leishmania MAP kinases. Binding affinity, ADME and toxicity of identified drug candidates were profiled. The anti-proliferative effects and mechanism of action were assessed in Leishmania, including changes in cell morphology, flagellar length, cell cycle progression, reactive oxygen species (ROS) generation, and intra-macrophage parasitic burden. KEY FINDINGS: 23 MAP kinases were identified from the Leishmania genome. Sorafenib and imatinib emerged as repurposable drug candidates and demonstrated excellent anti-proliferative effects in Leishmania. Treatment with these inhibitors resulted in significant changes in cell morphology, flagellar length, and cell cycle arrest. Furthermore, sorafenib and imatinib promoted ROS generation and reduced intra-macrophage parasitic burden, and elicited anti-leishmanial activity in in vivo experimental VL models. SIGNIFICANCE: Collectively, these results imply involvement of MAP kinases in infectivity and survival of the parasite and can pave the avenue for repurposing sorafenib and imatinib as anti-leishmanial agents. These findings contribute to the exploration of new treatment options for visceral leishmaniasis, particularly in the context of emerging drug resistance.

RAF1 facilitates KIT signaling and serves as a potential treatment target for gastrointestinal stromal tumor.

The aberrant activation of RAS/RAF/MEK/ERK signaling is important for KIT mutation-mediated tumorigenesis of gastrointestinal stromal tumor (GIST). In this study, we found that inhibition of RAF1 suppresses the activation of both wild-type KIT and primary KIT mutations in GIST, with primary KIT mutations showing greater sensitivity. This suggests a positive feedback loop between KIT and RAF1, wherein RAF1 facilitates KIT signaling. We further demonstrated that RAF1 associates with KIT and the kinase activity of RAF1 is necessary for its contribution to KIT activation. Accordingly, inhibition of RAF1 suppressed cell survival, proliferation, and cell cycle progression in vitro mediated by both wild-type KIT and primary KIT mutations. Inhibition of RAF1 in vivo suppressed GIST growth in a transgenic mouse model carrying germline KIT/V558A mutation, showing a similar treatment efficiency as imatinib, the first-line targeted therapeutic drug of GIST, while the combination use of imatinib and RAF1 inhibitor further suppressed tumor growth. Acquisition of drug-resistant secondary mutation of KIT is a major cause of treatment failure of GIST following targeted therapy. Like wild-type KIT and primary KIT mutations, inhibition of RAF1 suppressed the activation of secondary KIT mutation, and the cell survival, proliferation, cell cycle progression in vitro, and tumor growth in vivo mediated by secondary KIT mutation. However, the activation of secondary KIT mutation is less dependent on RAF1 compared with that of primary KIT mutations. Taken together, our results revealed that RAF1 facilitates KIT signaling and KIT mutation-mediated tumorigenesis of GIST, providing a rationale for further investigation into the use of RAF1 inhibitors alone or in combination with KIT inhibitor in the treatment of GIST, particularly in cases resistant to KIT inhibitors.

Role of CSF1R 550th-tryptophan in kusunokinin and CSF1R inhibitor binding and ligand-induced structural effect.

Binding affinity is an important factor in drug design to improve drug-target selectivity and specificity. In this study, in silico techniques based on molecular docking followed by molecular dynamics (MD) simulations were utilized to identify the key residue(s) for CSF1R binding affinity among 14 pan-tyrosine kinase inhibitors and 15 CSF1R-specific inhibitors. We found tryptophan at position 550 (W550) on the CSF1R binding site interacted with the inhibitors' aromatic ring in a π-π way that made the ligands better at binding. Upon W550-Alanine substitution (W550A), the binding affinity of trans-(-)-kusunokinin and imatinib to CSF1R was significantly decreased. However, in terms of structural features, W550 did not significantly affect overall CSF1R structure, but provided destabilizing effect upon mutation. The W550A also did not either cause ligand to change its binding site or conformational changes due to ligand binding. As a result of our findings, the π-π interaction with W550's aromatic ring could be still the choice for increasing binding affinity to CSF1R. Nevertheless, our study showed that the increasing binding to W550 of the design ligand may not ensure CSF1R specificity and inhibition since W550-ligand bound state did not induce significantly conformational change into inactive state.

The molecular basis of Abelson kinase regulation by its αI-helix.

Abelson tyrosine kinase (Abl) is regulated by the arrangement of its regulatory core, consisting sequentially of the SH3, SH2, and kinase (KD) domains, where an assembled or disassembled core corresponds to low or high kinase activity, respectively. It was recently established that binding of type II ATP site inhibitors, such as imatinib, generates a force from the KD N-lobe onto the SH3 domain and in consequence disassembles the core. Here, we demonstrate that the C-terminal αI-helix exerts an additional force toward the SH2 domain, which correlates both with kinase activity and type II inhibitor-induced disassembly. The αI-helix mutation E528K, which is responsible for the ABL1 malformation syndrome, strongly activates Abl by breaking a salt bridge with the KD C-lobe and thereby increasing the force onto the SH2 domain. In contrast, the allosteric inhibitor asciminib strongly reduces Abl's activity by fixating the αI-helix and reducing the force onto the SH2 domain. These observations are explained by a simple mechanical model of Abl activation involving forces from the KD N-lobe and the αI-helix onto the KD/SH2SH3 interface.

Involvement of BCR::ABL1 in laminin adhesion of Philadelphia chromosome-positive acute lymphoblastic leukemia through upregulation of integrin α6.

BACKGROUND: Adhesion of cancer cells to extracellular matrix laminin through the integrin superfamily reportedly induces drug resistance. Heterodimers of integrin α6 (CD49f) with integrin ß1 (CD29) or ß4 (CD104) are major functional receptors for laminin. Higher CD49f expression is reportedly associated with a poorer response to induction therapy in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Moreover, a xenograft mouse model transplanted with primary BCP-ALL cells revealed that neutralized antibody against CD49f improved survival after chemotherapy. AIMS: Considering the poor outcomes in Philadelphia chromosome (Ph)-positive ALL treated with conventional chemotherapy without tyrosine kinase inhibitors, we sought to investigate an involvement of the laminin adhesion. METHODS AND RESULTS: Ph-positive ALL cell lines expressed the highest levels of CD49f among the BCP-ALL cell lines with representative translocations, while CD29 and CD104 were ubiquitously expressed in BCP-ALL cell lines. The association of Ph-positive ALL with high levels of CD49f gene expression was also confirmed in two databases of childhood ALL cohorts. Ph-positive ALL cell lines attached to laminin and their laminin-binding properties were disrupted by blocking antibodies against CD49f and CD29 but not CD104. The cell surface expression of CD49f, but not CD29 and CD104, was downregulated by imatinib treatment in Ph-positive ALL cell lines, but not in their T315I-acquired sublines. Consistently, the laminin-binding properties were disrupted by the imatinib pre-treatment in the Ph-positive ALL cell line, but not in its T315I-acquired subline. CONCLUSION: BCR::ABL1 plays an essential role in the laminin adhesion of Ph-positive ALL cells through upregulation of CD49f.

Four and a half LIM domains 2 (FHL2) attenuates tumorigenesis of gastrointestinal stromal tumors (GISTs) by negatively regulating KIT signaling.

Gastrointestinal stromal tumors (GISTs) are predominately induced by KIT mutants. In this study, we found that four and a half LIM domains 2 (FHL2) was highly expressed in GISTs and KIT signaling dramatically increased FHL2 transcription while FHL2 inhibited KIT transcription. In addition, our results showed that FHL2 associated with KIT and increased the ubiquitination of both wild-type KIT and primary KIT mutants in GISTs, leading to decreased expression and activation of KIT although primary KIT mutants were less inhibited by FHL2 than wild-type KIT. In the animal experiments, loss of FHL2 expression in mice carrying germline KIT/V558A mutation which can develop GISTs resulted in increased tumor growth, but increased sensitivity of GISTs to imatinib treatment which is used as the first-line targeted therapy of GISTs, suggesting that FHL2 plays a role in the response of GISTs to KIT inhibitor. Unlike wild-type KIT and primary KIT mutants, we further found that FHL2 didn't alter the expression and activation of drug-resistant secondary KIT mutants. Taken together, our results indicated that FHL2 acts as the negative feedback of KIT signaling in GISTs while primary KIT mutants are less sensitive and secondary KIT mutants are resistant to the inhibition of FHL2.

Rapamycin vs TORin-1 or Gleevec vs Nilotinib: Simple chemical evolution that converts PAK1-blockers to TOR-blockers or vice versa?

Both PAK1 (RAC/CDC42-activating kinase 1) and TOR (Target of Rapamycin) are among the major oncogenic/ageing kinases. However, they play the opposite role in our immune system, namely immune system is suppressed by PAK1, while it requires TOR. Thus, PAK1-blockers, would be more effective for therapy of cancers, than TOR-blockers. Since 2015 when we discovered genetically that PDGF-induced melanogenesis depends on "PAK1", we are able to screening a series of PAK1-blockers as melanogenesis-inhibitors which could eventually promote longevity. Interestingly, rapamycin, the first TOR-inhibitor, promotes melanogenesis, clearly indicating that TOR suppresses melanogenesis. However, a new TOR-inhibitor called TORin-1 no longer suppresses immune system, and blocks melanogenesis in cell culture. These observations strongly indicate that TORin-1 acts as PAK1-blockers, instead of TOR-blockers, in vivo. Thus, it is most likely that melanogenesis in cell culture could enable us to discriminate PAK1-blockers from TORblockers.

High Level of CD8+PD-1+ Cells in Patients with Chronic Myeloid Leukemia Who Experienced Loss of MMR after Imatinib Discontinuation.

Treatment-free remission (TFR) is achieved in approximately half of chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors. The mechanisms responsible for TFR maintenance remain elusive. This study aimed to identify immune markers responsible for the control of residual CML cells early in the TFR (at 3 months), which may be the key to achieving long-term TFR and relapse-free survival (RFS) after discontinuation of imatinib. Our study included 63 CML patients after imatinib discontinuation, in whom comprehensive analysis of changes in the immune system was performed by flow cytometry, and changes in the BCR::ABL1 transcript levels were assessed by RQ-PCR and ddPCR. We demonstrated a significant increase in the percentage of CD8+PD-1+ cells in patients losing TFR. The level of CD8+PD-1+ cells is inversely related to the duration of treatment and incidence of deep molecular response (DMR) before discontinuation. Analysis of the ROC curve showed that the percentage of CD8+PD-1+ cells may be a significant factor in early molecular recurrence. Interestingly, at 3 months of TFR, patients with the e13a2 transcript had a significantly higher proportion of the PD-1-expressing immune cells compared to patients with the e14a2. Our results suggest the important involvement of CD8+PD-1+ cells in the success of TFR and may help in identifying a group of patients who could successfully discontinue imatinib.

Unveiling IL6R and MYC as Targeting Biomarkers in Imatinib-Resistant Chronic Myeloid Leukemia through Advanced Non-Invasive Apoptosis Detection Sensor Version 2 Detection.

The management of chronic myelogenous leukemia (CML) has seen significant progress with the introduction of tyrosine kinase inhibitors (TKIs), particularly Imatinib. However, a notable proportion of CML patients develop resistance to Imatinib, often due to the persistence of leukemia stem cells and resistance mechanisms independent of BCR::ABL1 This study investigates the roles of IL6R, IL7R, and MYC in Imatinib resistance by employing CRISPR/Cas9 for gene editing and the Non-Invasive Apoptosis Detection Sensor version 2 (NIADS v2) for apoptosis assessment. The results indicate that Imatinib-resistant K562 cells (K562-IR) predominantly express IL6R, IL7R, and MYC, with IL6R and MYC playing crucial roles in cell survival and sensitivity to Imatinib. Conversely, IL7R does not significantly impact cytotoxicity, either alone or in combination with Imatinib. Further genetic editing experiments confirm the protective functions of IL6R and MYC in K562-IR cells, suggesting their potential as therapeutic targets for overcoming Imatinib resistance in CML. This study contributes to understanding the mechanisms of Imatinib resistance in CML, proposing IL6R and MYC as pivotal targets for therapeutic strategies. Moreover, the utilization of NIADS v2 enhances our capability to analyze apoptosis and drug responses, contributing to a deeper understanding of CML pathogenesis and treatment options.

Deciphering the tumor immune microenvironment of imatinib-resistance in advanced gastrointestinal stromal tumors at single-cell resolution.

The heterogeneous nature of tumors presents a considerable obstacle in addressing imatinib resistance in advanced cases of gastrointestinal stromal tumors (GIST). To address this issue, we conducted single-cell RNA-sequencing in primary tumors as well as peritoneal and liver metastases from patients diagnosed with locally advanced or advanced GIST. Single-cell transcriptomic signatures of tumor microenvironment (TME) were analyzed. Immunohistochemistry and multiplex immunofluorescence staining were used to further validate it. This analysis revealed unique tumor evolutionary patterns, transcriptome features, dynamic cell-state changes, and different metabolic reprogramming. The findings indicate that in imatinib-resistant TME, tumor cells with activated immune and cytokine-mediated immune responses interacted with a higher proportion of Treg cells via the TIGIT-NECTIN2 axis. Future immunotherapeutic strategies targeting Treg may provide new directions for the treatment of imatinib-resistant patients. In addition, IDO1+ dendritic cells (DC) were highly enriched in imatinib-resistant TME, interacting with various myeloid cells via the BTLA-TNFRSF14 axis, while the interaction was not significant in imatinib-sensitive TME. Our study highlights the transcriptional heterogeneity and distinct immunosuppressive microenvironment of advanced GIST, which provides novel therapeutic strategies and innovative immunotherapeutic agents for imatinib resistance.

Comparison of the Efficacy Among Nilotinib, Dasatinib, Flumatinib and Imatinib in Newly Diagnosed Chronic-Phase Chronic Myeloid Leukemia Patients: A Real-World Multi-Center Retrospective Study.

BACKGROUND: There are limited data comprehensively comparing therapy responses and outcomes among nilotinib, dasatinib, flumatinib and imatinib for newly diagnosed chronic-phase chronic myeloid leukemia in a real-world setting. PATIENTS AND METHODS: Data from patients with chronic-phase CML receiving initial a second-generation tyrosine-kinase inhibitor (2G-TKI, nilotinib, dasatinib or flumatinib) or imatinib therapy from 77 Chinese centers were retrospectively interrogated. Propensity-score matching (PSM) analyses were performed to to compare therapy responses and outcomes among these 4 TKIs. RESULTS: 2,496 patients receiving initial nilotinib (n = 512), dasatinib (n = 134), flumatinib (n = 411) or imatinib (n = 1,439) therapy were retrospectively interrogated in this study. PSM analyses indicated that patients receiving initial nilotinib, dasatinib or flumatinib therapy had comparable cytogenetic and molecular responses (p = .28-.91) and survival outcomes including failure-free survival (FFS, p = .28-.43), progression-free survival (PFS, p = .19-.93) and overall survival (OS) (p values = .76-.78) but had significantly higher cumulative incidences of cytogenetic and molecular responses (all p values < .001) and higher probabilities of FFS (p < .001-.01) than those receiving imatinib therapy, despite comparable PFS (p = .18-.89) and OS (p = .23-.30). CONCLUSION: Nilotinib, dasatinib and flumatinib had comparable efficacy, and significantly higher therapy responses and higher FFS rates than imatinib in newly diagnosed CML patients. However, there were no significant differences in PFS and OS among these 4 TKIs. These real-world data may provide additional evidence for routine clinical assessments to identify more appropriate therapies.

Targeting mitochondrial bioenergetics by combination treatment with imatinib and dichloroacetate in human erythroleukemic K­562 and colorectal HCT­116 cancer cells.

Tumor malignant cells are characterized by dysregulation of mitochondrial bioenergetics due to the 'Warburg effect'. In the present study, this metabolic imbalance was explored as a potential target for novel cancer chemotherapy. Imatinib (IM) downregulates the expression levels of SCΟ2 and FRATAXIN (FXN) genes involved in the heme­dependent cytochrome c oxidase biosynthesis and assembly pathway in human erythroleukemic IM­sensitive K­562 chronic myeloid leukemia cells (K­562). In the present study, it was investigated whether the treatment of cancer cells with IM (an inhibitor of oxidative phosphorylation) separately, or together with dichloroacetate (DCA) (an inhibitor of glycolysis), can inhibit cell proliferation or cause death. Human K­562 and IM­chemoresistant K­562 chronic myeloid leukemia cells (K­562R), as well as human colorectal carcinoma cells HCT­116 (+/+p53) and (­/­p53, with double TP53 knock-in disruptions), were employed. Treatments of these cells with either IM (1 or 2 µM) and/or DCA (4 mΜ) were also assessed for the levels of several process biomarkers including SCO2, FXN, lactate dehydrogenase A, glyceraldehyde­3­phosphate dehydrogenase, pyruvate kinase M2, hypoxia inducing factor­1a, heme oxygenase­1, NF­κB, stem cell factor and vascular endothelial growth factor via western blot analysis. Computational network biology models were also applied to reveal the connections between the ten proteins examined. Combination treatment of IM with DCA caused extensive cell death (>75%) in K­562 and considerable (>45%) in HCT­116 (+/+p53) cultures, but less in K­562R and HCT­116 (­/­p53), with the latter deficient in full length p53 protein. Such treatment, markedly reduced reactive oxygen species levels, as measured by flow­cytometry, in K­562 cells and affected the oxidative phosphorylation and glycolytic biomarkers in all lines examined. These findings indicated, that targeting of cancer mitochondrial bioenergetics with such a combination treatment was very effective, although chemoresistance to IM in leukemia and the absence of a full length p53 in colorectal cells affected its impact.

Inhibitor Trapping in Kinases.

Recently, we identified a novel mechanism of enzyme inhibition in N-myristoyltransferases (NMTs), which we have named 'inhibitor trapping'. Inhibitor trapping occurs when the protein captures the small molecule within its structural confines, thereby preventing its free dissociation and resulting in a dramatic increase in inhibitor affinity and potency. Here, we demonstrate that inhibitor trapping also occurs in the kinases. Remarkably, the drug imatinib, which has revolutionized targeted cancer therapy, is entrapped in the structure of the Abl kinase. This effect is also observed in p38α kinase, where inhibitor trapping was found to depend on a 'magic' methyl group, which stabilizes the protein conformation and increases the affinity of the compound dramatically. Altogether, these results suggest that inhibitor trapping is not exclusive to N-myristoyltransferases, as it also occurs in the kinase family. Inhibitor trapping could enhance the binding affinity of an inhibitor by thousands of times and is as a key mechanism that plays a critical role in determining drug affinity and potency.

Establishment and characterization of NCC-DFSP5-C1: a novel patient-derived dermatofibrosarcoma protuberans cell line.

Dermatofibrosarcoma protuberans (DFSP) is the most prevalent dermal sarcoma, characterized by the presence of the fusion of the collagen type I alpha 1 (COL1A1) gene with the platelet-derived growth factor beta chain (PDGFB) gene. Although PDGF receptor inhibitor imatinib mesylate was approved for the treating patients with unresectable or metastatic DFSP, disease progression was shown in 9.2% of the patients. Therefore, developing novel therapeutic strategies is crucial for improving the prognosis of DFSP. Patient-derived cell lines play a vital role in preclinical studies; however, only a limited number of DFSP cell lines are currently available in public cell banks. Here, we successfully established a novel DFSP cell line (NCC-DFSP5-C1) using surgically resected tumor tissue from a patient with DFSP. NCC-DFSP5-C1 cells were confirmed to carry the COL1A1-PDGFB translocation and maintain the same mutation as the original tumor tissue. They exhibited consistent growth, formed spheroids, and were invasive. By screening a drug library using NCC-DFSP5-C1 and four previously established DFSP cell lines, we identified anti-cancer drugs that inhibit DFSP cell proliferation. Our observations suggest that the NCC-DFSP5-C1 cell line holds promise as a valuable tool for conducting fundamental and preclinical studies for DFSP.

KIT mutations and expression: current knowledge and new insights for overcoming IM resistance in GIST.

Gastrointestinal stromal tumor (GIST) is the most common sarcoma located in gastrointestinal tract and derived from the interstitial cell of Cajal (ICC) lineage. Both ICC and GIST cells highly rely on KIT signal pathway. Clinically, about 80-90% of treatment-naive GIST patients harbor primary KIT mutations, and special KIT-targeted TKI, imatinib (IM) showing dramatic efficacy but resistance invariably occur, 90% of them was due to the second resistance mutations emerging within the KIT gene. Although there are multiple variants of KIT mutant which did not show complete uniform biologic characteristics, most of them have high KIT expression level. Notably, the high expression level of KIT gene is not correlated to its gene amplification. Recently, accumulating evidences strongly indicated that the gene coding, epigenetic regulation, and pre- or post- protein translation of KIT mutants in GIST were quite different from that of wild type (WT) KIT. In this review, we elucidate the biologic mechanism of KIT variants and update the underlying mechanism of the expression of KIT gene, which are exclusively regulated in GIST, providing a promising yet evidence-based therapeutic landscape and possible target for the conquer of IM resistance. Video Abstract.