Small molecule somatostatin receptor subtype 4 (sst4) agonists are novel anti-inflammatory and analgesic drug candidates.

We provided strong proof of concept evidence that somatostatin mediates potent analgesic and anti-inflammatory actions via its receptor subtype 4 (sst4) located both at the periphery and the central nervous system. Therefore, sst4 agonists are promising novel drug candidates for neuropathic pain and neurogenic inflammation, but rational drug design was not possible due to the lack of knowledge about its 3-dimensional structure. We modeled the sst4 receptor structure, described its agonist binding properties, and characterized the binding of our novel small molecule sst4 agonists (4-phenetylamino-7H-pyrrolo[2,3-d]pyrimidine derivatives) using an in silico platform. In addition to the in silico binding data, somatostatin displacement by Compound 1 was demonstrated in the competitive binding assay on sst4-expressing cells. In vivo effects were investigated in rat models of neurogenic inflammation and chronic traumatic neuropathic pain. We defined high- and low-affinity binding pockets of sst4 for our ligands, binding of the highest affinity compounds were similar to that of the reference ligand J-2156. We showed potent G-protein activation with the highest potency of 10 nM EC50 value and highest efficacy of 342%. Oral administration of 100 µg/kg of 5 compounds significantly inhibited acute neurogenic plasma protein extravasation in the paw skin by 40-60%, one candidate abolished and 3 others diminished sciatic nerve-ligation induced neuropathic hyperalgesia by 28-62%. The in silico predictions on sst4-ligands were tested in biological systems. Low oral dose of our novel agonists inhibit neurogenic inflammation and neuropathic pain, which opens promising drug developmental perspectives for these unmet medical need conditions.

Targeted drug combination therapy design based on driver genes.

Targeted therapies against cancer types with more than one driver gene hold bright but elusive promise, since approved drugs are not available for all driver mutations and monotherapies often result in resistance. Targeting multiple driver genes in different pathways at the same time may provide an impact extensive enough to fight resistance. Our goal was to find synergistic drug combinations based on the availability of targeted drugs and their biological activity profiles and created an associated compound library based on driver gene-related protein targets. In this study, we would like to show that driver gene pattern based customized combination therapies are more effective than monotherapies on six cell lines and patient-derived primary cell cultures. We tested 55-102 drug combinations targeting driver genes and driver pathways for each cell line and found 25-85% of these combinations highly synergistic. Blocking 2-5 cancer pathways using only 2-3 targeted drugs was sufficient to reach high rates of tumor cell eradication at remarkably low concentrations. Our results demonstrate that the efficiency of cancer treatment may be significantly improved by combining drugs against multiple tumor specific drivers.

Regulation of influenza A virus mRNA splicing by CLK1.

Influenza A virus carries eight negative single-stranded RNAs and uses spliced mRNAs to increase the number of proteins produced from them. Several genome-wide screens for essential host factors for influenza A virus replication revealed a necessity for splicing and splicing-related factors, including Cdc-like kinase 1 (CLK1). This CLK family kinase plays a role in alternative splicing regulation through phosphorylation of serine-arginine rich (SR) proteins. To examine the influence that modulation of splicing regulation has on influenza infection, we analyzed the effect of CLK1 knockdown and inhibition. CLK1 knockdown in A549 cells reduced influenza A/WSN/33 virus replication and increased the level of splicing of segment 7, which encodes the viral M1 and M2 proteins. CLK1-/- mice infected with influenza A/England/195/2009 (H1N1pdm09) virus supported lower levels of virus replication than wild-type mice. Screening of newly developed CLK inhibitors revealed several compounds that have an effect on the level of splicing of influenza A gene segment M in different models and decrease influenza A/WSN/33 virus replication in A549 cells. The promising inhibitor KH-CB19, an indole-based enaminonitrile with unique binding mode for CLK1, and its even more selective analogue NIH39 showed high specificity towards CLK1 and had a similar effect on influenza mRNA splicing regulation. Taken together, our findings indicate that targeting host factors that regulate splicing of influenza mRNAs may represent a novel therapeutic approach.

Systematic Kinase Inhibitor Profiling Identifies CDK9 as a Synthetic Lethal Target in NUT Midline Carcinoma.

Kinase inhibitors represent the backbone of targeted cancer therapy, yet only a limited number of oncogenic drivers are directly druggable. By interrogating the activity of 1,505 kinase inhibitors, we found that BRD4-NUT-rearranged NUT midline carcinoma (NMC) cells are specifically killed by CDK9 inhibition (CDK9i) and depend on CDK9 and Cyclin-T1 expression. We show that CDK9i leads to robust induction of apoptosis and of markers of DNA damage response in NMC cells. While both CDK9i and bromodomain inhibition over time result in reduced Myc protein expression, only bromodomain inhibition induces cell differentiation and a p21-induced cell-cycle arrest in these cells. Finally, RNA-seq and ChIP-based analyses reveal a BRD4-NUT-specific CDK9i-induced perturbation of transcriptional elongation. Thus, our data provide a mechanistic basis for the genotype-dependent vulnerability of NMC cells to CDK9i that may be of relevance for the development of targeted therapies for NMC patients.

Molecular subtype specific efficacy of MEK inhibitors in pancreatic cancers.

Pancreatic cancer is an increasing cause of cancer related death worldwide. KRAS is the dominant oncogene in this cancer type and molecular rationale would indicate, that inhibitors of the downstream target MEK could be appropriate targeted agents, but clinical trials have failed so far to achieve statistically significant benefit in unselected patients. We aimed to identify predictive molecular biomarkers that can help to define subgroups where MEK inhibitors might be beneficial alone or in combination. Next-generation sequencing data of 50 genes in three pancreatic cancer cell lines (MiaPaCa2, BxPC3 and Panc1) were analyzed and compared to the molecular profile of 138 clinical pancreatic cancer samples to identify the molecular subtypes of pancreatic cancer these cell lines represent. Luminescent cell viability assay was used to determine the sensitivity of cell lines to kinase inhibitors. Western blot was used to analyze the pathway activity of the examined cell lines. According to our cell viability and pathway activity data on these model cell lines only cells harboring the rare G12C KRAS mutation and low EGFR expression are sensitive to single MEK inhibitor (trametinib) treatment. The common G12D KRAS mutation leads to elevated baseline Akt activity, thus treatment with single MEK inhibitors fails. However, combination of MEK and Akt inhibitors are synergistic in this case. In case of wild-type KRAS and high EGFR expression MEK inhibitor induced Akt phosphorylation leads to trametinib resistance which necessitates for MEK and EGFR or Akt inhibitor combination treatment. In all we provide strong preclinical rational and possible molecular mechanism to revisit MEK inhibitor therapy in pancreatic cancer in both monotherapy and combination, based on molecular profile analysis of pancreatic cancer samples and cell lines. According to our most remarkable finding, a small subgroup of patients with G12C KRAS mutation may still benefit from MEK inhibitor monotherapy.

Machine learning and docking models for Mycobacterium tuberculosis topoisomerase I.

There is a shortage of compounds that are directed towards new targets apart from those targeted by the FDA approved drugs used against Mycobacterium tuberculosis. Topoisomerase I (Mttopo I) is an essential mycobacterial enzyme and a promising target in this regard. However, it suffers from a shortage of known inhibitors. We have previously used computational approaches such as homology modeling and docking to propose 38 FDA approved drugs for testing and identified several active molecules. To follow on from this, we now describe the in vitro testing of a library of 639 compounds. These data were used to create machine learning models for Mttopo I which were further validated. The combined Mttopo I Bayesian model had a 5 fold cross validation receiver operator characteristic of 0.74 and sensitivity, specificity and concordance values above 0.76 and was used to select commercially available compounds for testing in vitro. The recently described crystal structure of Mttopo I was also compared with the previously described homology model and then used to dock the Mttopo I actives norclomipramine and imipramine. In summary, we describe our efforts to identify small molecule inhibitors of Mttopo I using a combination of machine learning modeling and docking studies in conjunction with screening of the selected molecules for enzyme inhibition. We demonstrate the experimental inhibition of Mttopo I by small molecule inhibitors and show that the enzyme can be readily targeted for lead molecule development.

The Inosine Monophosphate Dehydrogenase, GuaB2, Is a Vulnerable New Bactericidal Drug Target for Tuberculosis.

VCC234718, a molecule with growth inhibitory activity against Mycobacterium tuberculosis (Mtb), was identified by phenotypic screening of a 15344-compound library. Sequencing of a VCC234718-resistant mutant identified a Y487C substitution in the inosine monophosphate dehydrogenase, GuaB2, which was subsequently validated to be the primary molecular target of VCC234718 in Mtb. VCC234718 inhibits Mtb GuaB2 with a Ki of 100 nM and is uncompetitive with respect to IMP and NAD+. This compound binds at the NAD+ site, after IMP has bound, and makes direct interactions with IMP; therefore, the inhibitor is by definition uncompetitive. VCC234718 forms strong pi interactions with the Y487 residue side chain from the adjacent protomer in the tetramer, explaining the resistance-conferring mutation. In addition to sensitizing Mtb to VCC234718, depletion of GuaB2 was bactericidal in Mtb in vitro and in macrophages. When supplied at a high concentration (≥125 µM), guanine alleviated the toxicity of VCC234718 treatment or GuaB2 depletion via purine salvage. However, transcriptional silencing of guaB2 prevented Mtb from establishing an infection in mice, confirming that Mtb has limited access to guanine in this animal model. Together, these data provide compelling validation of GuaB2 as a new tuberculosis drug target.

Identification of aminopyrimidine-sulfonamides as potent modulators of Wag31-mediated cell elongation in mycobacteria.

There is an urgent need to discover new anti-tubercular agents with novel mechanisms of action in order to tackle the scourge of drug-resistant tuberculosis. Here, we report the identification of such a molecule - an AminoPYrimidine-Sulfonamide (APYS1) that has potent, bactericidal activity against M. tuberculosis. Mutations in APYS1-resistant M. tuberculosis mapped exclusively to wag31, a gene that encodes a scaffolding protein thought to orchestrate cell elongation. Recombineering confirmed that a Gln201Arg mutation in Wag31 was sufficient to cause resistance to APYS1, however, neither overexpression nor conditional depletion of wag31 impacted M. tuberculosis susceptibility to this compound. In contrast, expression of the wildtype allele of wag31 in APYS1-resistant M. tuberculosis was dominant and restored susceptibility to APYS1 to wildtype levels. Time-lapse imaging and scanning electron microscopy revealed that APYS1 caused gross malformation of the old pole of M. tuberculosis, with eventual lysis. These effects resembled the morphological changes observed following transcriptional silencing of wag31 in M. tuberculosis. These data show that Wag31 is likely not the direct target of APYS1, but the striking phenotypic similarity between APYS1 exposure and genetic depletion of Wag31 in M. tuberculosis suggests that APYS1 might indirectly affect Wag31 through an as yet unknown mechanism.

Development of a 3D Tissue Culture-Based High-Content Screening Platform That Uses Phenotypic Profiling to Discriminate Selective Inhibitors of Receptor Tyrosine Kinases.

3D tissue cultures provide a more physiologically relevant context for the screening of compounds, compared with 2D cell cultures. Cells cultured in 3D hydrogels also show complex phenotypes, increasing the scope for phenotypic profiling. Here we describe a high-content screening platform that uses invasive human prostate cancer cells cultured in 3D in standard 384-well assay plates to study the activity of potential therapeutic small molecules and antibody biologics. Image analysis tools were developed to process 3D image data to measure over 800 phenotypic parameters. Multiparametric analysis was used to evaluate the effect of compounds on tissue morphology. We applied this screening platform to measure the activity and selectivity of inhibitors of the c-Met and epidermal growth factor (EGF) receptor (EGFR) tyrosine kinases in 3D cultured prostate carcinoma cells. c-Met and EGFR activity was quantified based on the phenotypic profiles induced by their respective ligands, hepatocyte growth factor and EGF. The screening method was applied to a novel collection of 80 putative inhibitors of c-Met and EGFR. Compounds were identified that induced phenotypic profiles indicative of selective inhibition of c-Met, EGFR, or bispecific inhibition of both targets. In conclusion, we describe a fully scalable high-content screening platform that uses phenotypic profiling to discriminate selective and nonselective (off-target) inhibitors in a physiologically relevant 3D cell culture setting.

Synthesis, characterization and systematic comparison of FITC-labelled GnRH-I, -II and -III analogues on various tumour cells.

Targeted tumour therapy is the focus of recent cancer research. Gonadotropin-releasing hormone (GnRH) analogues are able to deliver anticancer agents selectively into tumour cells, which highly express GnRH receptors. However, the effectiveness of different analogues as targeting moiety in drug delivery systems is rarely compared, and the investigated types of cancer are also limited. Therefore, we prepared selectively labelled, fluorescent derivatives of GnRH-I, -II and -III analogues, which were successfully used for drug targeting. In this manuscript, we investigated these analogues' solubility, stability and passive membrane permeability and compared their cellular uptake by various cancer cells. We found that these labelled GnRH conjugates provide great detectability, without undesired cytotoxicity and passive membrane permeability. The introduced experiments with these conjugates proved their reliable tracking, quantification and comparison. Cellular uptake efficiency was studied on human breast, colon, pancreas and prostate cancer cells (MCF-7, HT-29, BxPC-3, LNCaP) and on dog kidney cells (Madin-Darby canine kidney). Each of the three conjugates was taken up by GnRH-I receptor-expressing cells, but the different cells preferred different analogues. Furthermore, we demonstrated for the first time the high cell surface expression of GnRH-I receptors and the effective cellular uptake of GnRH analogues on human pharynx tumour (Detroit-562) cells. In summary, our presented results detail that the introduced conjugates could be innovative tools for the examination of the GnRH-based drug delivery systems on various cells and offer novel information about these peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Design and synthesis of new imidazo[1,2-a]pyridine and imidazo[1,2-a]pyrazine derivatives with antiproliferative activity against melanoma cells.

Melanoma is an aggressive form of skin cancer and it is generally associated with poor prognosis in patients with late-stage disease. Due to the increasing occurrence of melanoma, there is a need for the development of novel therapies. A new series of diarylamide and diarylurea derivatives containing imidazo[1,2-a]pyridine or imidazo[1,2-a]pyrazine scaffold was designed and synthesized to investigate their in vitro efficacy against the A375P human melanoma cell line. We found several compounds expressing submicromolar IC50 values against the A375P cells, from which 15d, 17e, 18c, 18h, 18i demonstrated the highest potencies with IC50 below 0.06 µM.

Novel compounds reducing IRS-1 serine phosphorylation for treatment of diabetes.

Activation of various interacting stress kinases, particularly the c-Jun N-terminal kinases (JNK), and a concomitant phosphorylation of insulin receptor substrate 1 (IRS-1) at serine 307 play a central role both in insulin resistance and in ß-cell dysfunction. IRS-1 phosphorylation is stimulated by elevated free fatty acid levels through different pathways in obesity. A series of novel pyrido[2,3-d]pyrimidin-7-one derivatives were synthesized as potential antidiabetic agents, preventing IRS-1 phosphorylation at serine 307 in a cellular model of lipotoxicity and type 2 diabetes.

Small Molecule Inhibition of ERK Dimerization Prevents Tumorigenesis by RAS-ERK Pathway Oncogenes.

Nearly 50% of human malignancies exhibit unregulated RAS-ERK signaling; inhibiting it is a valid strategy for antineoplastic intervention. Upon activation, ERK dimerize, which is essential for ERK extranuclear, but not for nuclear, signaling. Here, we describe a small molecule inhibitor for ERK dimerization that, without affecting ERK phosphorylation, forestalls tumorigenesis driven by RAS-ERK pathway oncogenes. This compound is unaffected by resistance mechanisms that hamper classical RAS-ERK pathway inhibitors. Thus, ERK dimerization inhibitors provide the proof of principle for two understudied concepts in cancer therapy: (1) the blockade of sub-localization-specific sub-signals, rather than total signals, as a means of impeding oncogenic RAS-ERK signaling and (2) targeting regulatory protein-protein interactions, rather than catalytic activities, as an approach for producing effective antitumor agents.

Decreased functional activity of multidrug resistance protein in primary colorectal cancer.

BACKGROUND: The ATP-Binding Cassette (ABC)-transporter MultiDrug Resistance Protein 1 (MDR1) and Multidrug Resistance Related Protein 1 (MRP1) are expressed on the surface of enterocytes, which has led to the belief that these high capacity transporters are responsible for modulating chemosensitvity of colorectal cancer. Several immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) studies have provided controversial results in regards to the expression levels of these two ABC-transporters in colorectal cancer. Our study was designed to determine the yet uninvestigated functional activity of MDR1 and MRP1 transporters in normal human enterocytes compared to colorectal cancer cells from surgical biopsies. METHODS: 100 colorectal cancer and 28 adjacent healthy mucosa samples were obtained by intraoperative surgical sampling. Activity of MDR1 and MRP1 of viable epithelial and cancer cells were determined separately with the modified calcein-assay for multidrug resistance activity and sufficient data of 73 cancer and 11 healthy mucosa was analyzed statistically. RESULTS: Significantly decreased mean MDR1 activity was found in primary colorectal cancer samples compared to normal mucosa, while mean MRP1 activity showed no significant change. Functional activity was not affected by gender, age, stage or grade and localization of the tumor. CONCLUSION: We found lower MDR activity in cancer cells versus adjacent, apparently, healthy control tissue, thus, contrary to general belief, MDR activity seems not to play a major role in primary drug resistance, but might rather explain preferential/selective activity of Irinotecan and/or Oxaliplatin. Still, this picture might be more complex since chemotherapy by itself might alter MDR activity, and furthermore, today limited data is available about MDR activity of cancer stem cells in colorectal cancers. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1675739129145824.

Targeting vascular endothelial growth factor receptor 2 and protein kinase D1 related pathways by a multiple kinase inhibitor in angiogenesis and inflammation related processes in vitro.

Emerging evidence suggests that the vascular endothelial growth factor receptor 2 (VEGFR2) and protein kinase D1 (PKD1) signaling axis plays a critical role in normal and pathological angiogenesis and inflammation related processes. Despite all efforts, the currently available therapeutic interventions are limited. Prior studies have also proved that a multiple target inhibitor can be more efficient compared to a single target one. Therefore, development of novel inflammatory pathway-specific inhibitors would be of great value. To test this possibility, we screened our molecular library using recombinant kinase assays and identified the previously described compound VCC251801 with strong inhibitory effect on both VEGFR2 and PKD1. We further analyzed the effect of VCC251801 in the endothelium-derived EA.hy926 cell line and in different inflammatory cell types. In EA.hy926 cells, VCC251801 potently inhibited the intracellular activation and signaling of VEGFR2 and PKD1 which inhibition eventually resulted in diminished cell proliferation. In this model, our compound was also an efficient inhibitor of in vitro angiogenesis by interfering with endothelial cell migration and tube formation processes. Our results from functional assays in inflammatory cellular models such as neutrophils and mast cells suggested an anti-inflammatory effect of VCC251801. The neutrophil study showed that VCC251801 specifically blocked the immobilized immune-complex and the adhesion dependent TNF-α -fibrinogen stimulated neutrophil activation. Furthermore, similar results were found in mast cell degranulation assay where VCC251801 caused significant reduction of mast cell response. In summary, we described a novel function of a multiple kinase inhibitor which strongly inhibits the VEGFR2-PKD1 signaling and might be a novel inhibitor of pathological inflammatory pathways.

Gremlin regulates renal inflammation via the vascular endothelial growth factor receptor 2 pathway.

Inflammation is a main feature of progressive kidney disease. Gremlin binds to bone morphogenetic proteins (BMPs), acting as an antagonist and regulating nephrogenesis and fibrosis among other processes. Gremlin also binds to vascular endothelial growth factor receptor-2 (VEGFR2) in endothelial cells to induce angiogenesis. In renal cells, gremlin regulates proliferation and fibrosis, but there are no data about inflammatory-related events. We have investigated the direct effects of gremlin in the kidney, evaluating whether VEGFR2 is a functional gremlin receptor. Administration of recombinant gremlin to murine kidneys induced rapid and sustained activation of VEGFR2 signalling, located in proximal tubular epithelial cells. Gremlin bound to VEGFR2 in these cells in vitro, activating this signalling pathway independently of its action as an antagonist of BMPs. In vivo, gremlin caused early renal damage, characterized by activation of the nuclear factor (NF)-κB pathway linked to up-regulation of pro-inflammatory factors and infiltration of immune inflammatory cells. VEGFR2 blockade diminished gremlin-induced renal inflammatory responses. The link between gremlin/VEGFR2 and NF-κB/inflammation was confirmed in vitro. Gremlin overexpression was associated with VEGFR2 activation in human renal disease and in the unilateral ureteral obstruction experimental model, where VEGFR2 kinase inhibition diminished renal inflammation. Our data show that a gremlin/VEGFR2 axis participates in renal inflammation and could be a novel target for kidney disease.

Lead selection and characterization of antitubercular compounds using the Nested Chemical Library.

Discovering new drugs to treat tuberculosis more efficiently and to overcome multidrug resistance is a world health priority. To find novel antitubercular agents several approaches have been used in various institutions worldwide, including target-based approaches against several validated mycobacterial enzymes and phenotypic screens. We screened more than 17,000 compounds from Vichem's Nested Chemical Library™ using an integrated strategy involving whole cell-based assays with Corynebacterium glutamicum and Mycobacterium tuberculosis, and target-based assays with protein kinases PknA, PknB and PknG as well as other targets such as PimA and bacterial topoisomerases simultaneously. With the help of the target-based approach we have found very potent hits inhibiting the selected target enzymes, but good minimal inhibitory concentrations (MIC) against M. tuberculosis were not achieved. Focussing on the whole cell-based approach several potent hits were found which displayed minimal inhibitory concentrations (MIC) against M. tuberculosis below 10 µM and were non-mutagenic, non-cytotoxic and the targets of some of the hits were also identified. The most active hits represented various scaffolds. Medicinal chemistry-based lead optimization was performed applying various strategies and, as a consequence, a series of novel potent compounds were synthesized. These efforts resulted in some effective potential antitubercular lead compounds which were confirmed in phenotypic assays.

[Pyrido[2,3-b]pyrazines inhibiting both erlotinib-sensitive and erlotinib-resistant cell lines, and their preparation via regioselective condensation reaction]. / Erlotinib-érzékeny és erlotinib-rezisztens sejtvonalakat gátló pirido[2,3-b] pirazinok, és eloállításuk régiószelektív kondenzációs reakcióval.

The EGFR inhibitor erlotinib possesses high anti-tumor effect but despite the good clinical responses in most of the cases recrudescence occures. This can be attributed to a secondary, acquired mutation causing resistance to tyrosine kinase inhibitors. In our work we were looking for small-molecule inhibitors, which simultaneously affect on the proliferation of erlotinib-sensitive PC9 cells and PC9-ER erlotinib-resistant cells. A set of molecules were selected from Vichem Chemie Research Ltd.'s kinase inhibitor compound library (Nested Chemical Library™). According to the results of medium throughput screening (MTS) of this set of compounds, novel structures with pyrido[2,3-b]pyrazine core were designed. These compounds were proved to be effective inhibitors of resistant cells in phenotypic screening. Based on these results structure-activity relationships were set up. The pyrido[2,3-b]pyrazine core was synthesized by a condensation reaction, which resulting two asymmetric products. In the reaction two regioisomer intermediates formed, and one of the products is the intermediate of the effective compounds. This condensation reaction was optimized, the regioisomers were identified by NMR analysis and X-ray crystallography. As a result of optimization we found that lower reaction temperature and replacement of dimethylformamide solvent with trifluoroacetic acid provided the undesired isomer in less than 2 % ratio.

Combined inhibition of AXL, Lyn and p130Cas kinases block migration of triple negative breast cancer cells.

Blocking the migration of metastatic cancer cells is a major goal in the therapy of cancer. The receptor tyrosine kinase AXL is one of the main triggers for cancer cell migration in neoplasia of breast, colon, skin, thyroid and prostate. In our study we analyzed the effect of AXL inhibition on cell motility and viability in triple negative breast cancer cell lines overexpressing AXL. Thereby we reveal that the compound BMS777607, exhibiting the lowest IC50 values for inhibition of AXL kinase activity in the studied cell lines, attenuates cell motility to a lower extent than the kinase inhibitors MPCD84111 and SKI606. By analyzing the target kinases of MPCD84111 and SKI606 with kinase profiling assays we identified Lyn, a Src family kinase, as a target of both compounds. Knockdown of Lyn and the migration-related CRK-associated substrate (p130Cas), had a significant inhibitory effect on cell migration. Taken together, our findings highlight the importance of combinatorial or multikinase inhibition of non-receptor tyrosine kinases and AXL receptor tyrosine kinase in the therapy of triple negative breast cancer.