Persistent Cancer Cells: The Deadly Survivors.

Persistent cancer cells are the discrete and usually undetected cells that survive cancer drug treatment and constitute a major cause of treatment failure. These cells are characterized by their slow proliferation, highly flexible energy consumption, adaptation to their microenvironment, and phenotypic plasticity. Mechanisms that underlie their persistence offer highly coveted and sought-after therapeutic targets, and include diverse epigenetic, transcriptional, and translational regulatory processes, as well as complex cell-cell interactions. Although the successful clinical targeting of persistent cancer cells remains to be realized, immense progress has been made in understanding their persistence, yielding promising preclinical results.

Resistance of HNSCC cell models to pan-FGFR inhibition depends on the EMT phenotype associating with clinical outcome.

BACKGROUND: Focal adhesion signaling involving receptor tyrosine kinases (RTK) and integrins co-controls cancer cell survival and therapy resistance. However, co-dependencies between these receptors and therapeutically exploitable vulnerabilities remain largely elusive in HPV-negative head and neck squamous cell carcinoma (HNSCC). METHODS: The cytotoxic and radiochemosensitizing potential of targeting 10 RTK and ß1 integrin was determined in up to 20 3D matrix-grown HNSCC cell models followed by drug screening and patient-derived organoid validation. RNA sequencing and protein-based biochemical assays were performed for molecular characterization. Bioinformatically identified transcriptomic signatures were applied to patient cohorts. RESULTS: Fibroblast growth factor receptor (FGFR 1-4) targeting exhibited the strongest cytotoxic and radiosensitizing effects as monotherapy and combined with ß1 integrin inhibition, exceeding the efficacy of the other RTK studied. Pharmacological pan-FGFR inhibition elicited responses ranging from cytotoxicity/radiochemosensitization to resistance/radiation protection. RNA sequence analysis revealed a mesenchymal-to-epithelial transition (MET) in sensitive cell models, whereas resistant cell models exhibited a partial epithelial-to-mesenchymal transition (EMT). Accordingly, inhibition of EMT-associated kinases such as EGFR caused reduced adaptive resistance and enhanced (radio)sensitization to FGFR inhibition cell model- and organoid-dependently. Transferring the EMT-associated transcriptomic profiles to HNSCC patient cohorts not only demonstrated their prognostic value but also provided a conclusive validation of the presence of EGFR-related vulnerabilities that can be strategically exploited for therapeutic interventions. CONCLUSIONS: This study demonstrates that pan-FGFR inhibition elicits a beneficial radiochemosensitizing and a detrimental radioprotective potential in HNSCC cell models. Adaptive EMT-associated resistance appears to be of clinical importance, and we provide effective molecular approaches to exploit this therapeutically.

SIRT7 promotes mitochondrial biogenesis to render the adaptive resistance to MAPK inhibition in melanoma.

Melanoma, arising from the malignant transformation of melanocytes, stands as the most lethal type of skin cancer. While significant strides have been made in targeted therapy and immunotherapy, substantially enhancing therapeutic efficacy, the prognosis for melanoma patients remains unoptimistic. SIRT7, a nuclear-localized deacetylase, plays a pivotal role in maintaining cellular homeostasis and adapting to external stressors in melanoma, with its activity closely tied to intracellular nicotinamide adenine dinucleotide (NAD+). However, its involvement in adaptive resistance to targeted therapy remains unclear. Herein, we unveil that up-regulated SIRT7 promotes mitochondrial biogenesis to render the adaptive resistance to MAPK inhibition in melanoma. Initially, we observed a significant increase of SIRT7 expression in publicly available datasets following targeted therapy within a short duration. In consistent, we found elevated SIRT7 expression in melanoma cells subjected to BRAF or MEK inhibitors in vitro. The up-regulation of SIRT7 expression was also confirmed in xenograft tumors in mice after targeted therapy in vivo. Furthermore, we proved that SIRT7 deficiency led to decreased cell viability upon prolonged exposure to BRAF or MEK inhibitors, accompanied by an increase in cell apoptosis. Mechanistically, SIRT7 deficiency restrained the upregulation of genes associated with mitochondrial biogenesis and intracellular ATP levels in response to targeted therapy treatment in melanoma cells. Ultimately, we proved that SIRT7 deficieny could sensitize BRAF-mutant melanoma cells to MAPK inhibition targeted therapy in vivo. In conclusion, our findings underscore the role of SIRT7 in fostering adaptive resistance to targeted therapy through the facilitation of mitochondrial biogenesis. Targeting SIRT7 emerges as a promising strategy to overcome MAPK inhibitor adaptive resistance in melanoma.

Increased resistance against tellurite is conferred by a mutation in the promoter region of uncommon tellurite resistance gene tehB in the ter-negative Shiga toxin-producing Escherichia coli O157:H7.

Resistance to potassium tellurite (PT) is an important indicator in isolating Shiga toxin-producing Escherichia coli (STEC) O157:H7 and other major STEC serogroups. Common resistance determinant genes are encoded in the ter gene cluster. We found an O157:H7 isolate that does not harbor ter but is resistant to PT. One nonsynonymous mutation was found in another PT resistance gene, tehA, through whole-genome sequence analyses. To elucidate the contribution of this mutation to PT resistance, complementation of tehA and the related gene tehB in isogenic strains and quantitative RT‒PCR were performed. The results indicated that the point mutation not only changed an amino acid of tehA, but also was positioned on a putative internal promoter of tehB and increased PT resistance by elevating tehB mRNA expression. Meanwhile, the amino acid change in tehA had negligible impact on the PT resistance. Comprehensive screening revealed that 2.3% of O157:H7 isolates in Japan did not harbor the ter gene cluster, but the same mutation in tehA was not found. These results suggested that PT resistance in E. coli can be enhanced through one mutational event even in ter-negative strains. IMPORTANCE: Selective agents are important for isolating Shiga toxin-producing Escherichia coli (STEC) because the undesirable growth of microflora should be inhibited. Potassium tellurite (PT) is a common selective agent for major STEC serotypes. In this study, we found a novel variant of PT resistance genes, tehAB, in STEC O157:H7. Molecular experiments clearly showed that one point mutation in a predicted internal promoter region of tehB upregulated the expression of the gene and consequently led to increased resistance to PT. Because tehAB genes are ubiquitous across E. coli, these results provide universal insight into PT resistance in this species.

YAP inhibition overcomes adaptive resistance in HER2-positive gastric cancer treated with trastuzumab via the AKT/mTOR and ERK/mTOR axis.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2)-positive gastric cancer (GC) is a heterogeneous GC subtype characterized by the overexpression of HER2. To date, few specific targeted therapies have demonstrated durable efficacy in HER2-positive GC patients, with resistance to trastuzumab typically emerging within 1 year. However, the mechanisms of resistance to trastuzumab remain incompletely understood, presenting a significant challenge to clinical practice. METHODS: In this study, we integrated genetic screening and bulk transcriptome and epigenomic profiling to define the mechanisms mediating adaptive resistance to HER2 inhibitors and identify potential effective therapeutic strategies for treating HER2-positive GCs. RESULTS: We revealed a potential association between adaptive resistance to trastuzumab in HER2-positive GC and the expression of YES-associated protein (YAP). Notably, our investigation revealed that long-term administration of trastuzumab triggers extensive chromatin remodeling and initiates YAP gene transcription in HER2-positive cells characterized by the initial inhibition and subsequent reactivation. Furthermore, treatment of HER2-positive GC cells and cell line-derived xenografts (CDX) models with YAP inhibitors in combination with trastuzumab was found to induce synergistic effects through the AKT/mTOR and ERK/mTOR pathways. CONCLUSION: These findings underscore the pivotal role of reactivated YAP and mTOR signaling pathways in the development of adaptive resistance to trastuzumab and may serve as a promising joint target to overcome resistance to trastuzumab.

Adaptive chromatin remodeling and transcriptional changes of the functional kinome in tumor cells in response to targeted kinase inhibition.

Pharmacological inhibition of protein kinases induces adaptive reprogramming of tumor cell regulatory networks by altering expression of genes that regulate signaling, including protein kinases. Adaptive responses are dependent on transcriptional changes resulting from remodeling of enhancer and promoter landscapes. Enhancer and promoter remodeling in response to targeted kinase inhibition is controlled by changes in open chromatin state and by activity of specific transcription factors, such as c-MYC. This review focuses on the dynamic plasticity of protein kinase expression of the tumor cell kinome and the resulting adaptive resistance to targeted kinase inhibition. Plasticity of the functional kinome has been shown in patient window trials where triple-negative and human epidermal growth factor receptor 2-positive breast cancer patient tumors were characterized by RNAseq after biopsies before and after 1 week of therapy. The expressed kinome changed dramatically during drug treatment, and these changes in kinase expression were shown in cell lines and xenografts in mice to be correlated with adaptive tumor cell drug resistance. The dynamic transcriptional nature of the kinome also differs for inhibitors targeting different kinase signaling pathways (e.g., BRAF-MEK-ERK versus PI3K-AKT) that are commonly activated in cancers. Heterogeneity arising from differences in gene regulation and mutations represents a challenge to therapeutic durability and prevention of clinical drug resistance with drug-tolerant tumor cell populations developing and persisting through treatment. We conclude that understanding the heterogeneity of kinase expression at baseline and in response to therapy is imperative for development of combinations and timing intervals of therapies making interventions durable.

Characterization of Pseudomonas aeruginosa resistance to ceftolozane-tazobactam due to ampC and/or ampD mutations observed during treatment using semi-mechanistic PKPD modeling.

A double ampC (AmpCG183D) and ampD (AmpDH157Y) genes mutations have been identified by whole genome sequencing in a Pseudomonas aeruginosa (PaS) that became resistant (PaR) in a patient treated by ceftolozane/tazobactam (C/T). To precisely characterize the respective contributions of these mutations on the decreased susceptibility to C/T and on the parallel increased susceptibility to imipenem (IMI), mutants were generated by homologous recombination in PAO1 reference strain (PAO1- AmpCG183D, PAO1-AmpDH157Y, PAO1-AmpCG183D/AmpDH157Y) and in PaR (PaR-AmpCPaS/AmpDPaS). Sequential time-kill curve experiments were conducted on all strains and analyzed by semi-mechanistic PKPD modeling. A PKPD model with adaptation successfully described the data, allowing discrimination between initial and time-related (adaptive resistance) effects of mutations. With PAO1 and mutant-derived strains, initial EC50 values increased by 1.4, 4.1, and 29-fold after AmpCG183D , AmpDH157Y and AmpCG183D/AmpDH157Y mutations, respectively. EC50 values were increased by 320, 12.4, and 55-fold at the end of the 2 nd experiment. EC50 of PAO1-AmpCG183D/AmpDH157Y was higher than that of single mutants at any time of the experiments. Within the PaR clinical background, reversal of AmpCG183D, and AmpDH157Y mutations led to an important decrease of EC50 value, from 80.5 mg/L to 6.77 mg/L for PaR and PaR-AmpCPaS/AmpDPaS, respectively. The effect of mutations on IMI susceptibility mainly showed that the AmpCG183D mutation prevented the emergence of adaptive resistance. The model successfully described the separate and combined effect of AmpCG183D and AmpDH157Y mutations against C/T and IMI, allowing discrimination and quantification of the initial and time-related effects of mutations. This method could be reproduced in clinical strains to decipher complex resistance mechanisms.

Novel Cocktail Therapy Based on a Nanocarrier with an Efficient Transcytosis Property Reverses the Dynamically Deteriorating Tumor Microenvironment for Enhanced Immunotherapy.

The immune checkpoint blockade (ICB) faces a low response rate in clinical cancer treatment. Chemotherapy could enhance the response rate of the ICB, but patients would suffer from side effects. The off-target toxicity could be reduced by loading the chemotherapeutic agent through nanocarriers. Therefore, we developed a polymeric carrier for doxorubicin (DOX) loading to form DOX nanoparticles (DOX NPs), which were spatiotemporally responsive to the tumor microenvironment (TME). DOX NPs had an efficient transcytosis property for deep tumor infiltration and sustained drug release ability. Unfortunately, a binary therapy of DOX NPs and ICB induces tumor adaptive resistance and causes dynamic deterioration of the TME. We propose for the first time that TGF-ß1 is a major cause of tumor adaptive resistance and developed an immune cocktail therapy containing DOX NPs, ICB, and TGF-ß1 gene silencing nanoparticles. This therapy successfully overcame tumor adaptive resistance by reversing the immunosuppressive TME and achieved enhanced tumor treatment efficiency.

Inhibition of the DAPKs-L13a axis prevents a GAIT-like motif-mediated HuR insufficiency in melanoma cells.

We previously showed that the adaptive response of BRAFV600-mutated melanoma cells to BRAF inhibition emerges from a subpopulation of cells expressing an intermittent lower level of the mRNA-binding protein HuR. In this study, following initial overexpression experiments in which we confirm our previous results, we use wild-type and mutants HuR full-length mRNA constructs and in vivo-interacting assays and demonstrate that a highly conserved interferon-γ-activated inhibitor of translation (GAIT)-like motif located upstream of the GU-rich elements of HuR major polyadenylation site (PAS2), interacts with constituents of the GAIT complex and affects HuR post-transcriptional expression regulation. Knockdown of the ribosomal protein L13a or the inhibition of the DAPK1-ZIPK axis involved in L13a phosphorylation, reduces the proportion of HuRLow cells at steady-state and attenuates the adaptive response of the whole melanoma-cell population to BRAF inhibition. These results have further potential therapeutic implications for disease conditions associated with HuR insufficient expression.

Characterization of anticancer drug resistance by reverse-phase protein array: new targets and strategies.

INTRODUCTION: Drug resistance is the main barrier to achieving cancer cures with medical therapy. Cancer drug resistance occurs, in part, due to adaptation of the tumor and microenvironment to therapeutic stress at a proteomic level. Reverse-phase protein arrays (RPPA) are well suited to proteomic analysis of drug resistance due to high sample throughput, sensitive detection of phosphoproteins, and validation for a large number of critical cellular pathways. AREAS COVERED: This review summarizes contributions of RPPA to understanding and combating drug resistance. In particular, contributions of RPPA to understanding resistance to PARP inhibitors, BRAF inhibitors, immune checkpoint inhibitors, and breast cancer investigational therapies are discussed. Articles reviewed were identified by MEDLINE, Scopus, and Cochrane search for keywords 'proteomics,' 'reverse-phase protein array,' 'drug resistance,' 'PARP inhibitor,' 'BRAF inhibitor,' 'immune checkpoint inhibitor,' and 'I-SPY' spanning October 1, 1960 - October 1, 2021. EXPERT OPINION: Precision oncology has thus far failed to convert the armament of targeted therapies into durable responses for most patients, highlighting that genetic sequencing alone is insufficient to guide therapy selection and overcome drug resistance. Combined genomic and proteomic analyses paired with creative drug combinations and dosing strategies hold promise for maturing precision oncology into an era of improved patient outcomes.

Metabolomics reveals the mechanism of Antarctic yeast Rhodotorula mucliaginosa AN5 to cope with cadmium stress.

Heavy metal pollution in Antarctica has far exceeded expectations. Antarctic yeast is widely present in polar marine environment. The mechanisms of metabolomics effect of heavy metal on polar yeast have not been reported previously. In this study, gas chromatography-mass spectrometry (GC-MS) wascarried out to performed the metabolite profiling analysis of Antarctic sea-ice yeast Rhodotorula mucilaginosa AN5 exposed to different cadmium (Cd) stresses of 5 mM (HM5), 10 mM (HM10) and 20 mM (HM20), respectively. Metabolic profile analysis showed that the composition and contents of cellular metabolites have been altered by cadmium. 93 different metabolites were identified altogether, among which 23, 58 and 81 different metabolites were found in HM5, HM10 and HM20 group respectively. MetaboAnalyst analysis showed that in HM5, HM10 and HM20 groups, 12, 24 and 31 metabolic pathways were involved in the stress of cadmium to R. mucilaginosa, respectively. By contrasting with Kyoto Encyclopedia of Genes and Genomes database, we discovered that exposure of yeast AN5 to Cd stress resulted in profound biochemical changes including amino acids, organic acids and saccharides. These results will supply a nonnegligible basis of studying the adaptive resistance mechanism of Antarctic yeast Rhodotorula mucilaginosa to heavy metal.

Cholesterol Regulates the Tumor Adaptive Resistance to MAPK Pathway Inhibition.

Although targeted MAPK pathway inhibition has achieved remarkable patient responses in many cancers, the development of resistance has remained a critical challenge. Adaptive tumor response underlies the drug resistance. Furthermore, such bypass mechanisms often lead to the activation of many pro-survival kinases, which complicates the rational design of combination therapies. Here, we performed global tyrosine phosphoproteomic (pTyr) analyses and demonstrated that targeted MAPK signaling inhibition in melanoma leads to a profound remodeling of the pTyr proteome. Intriguingly, altered cholesterol metabolism might drive, in a coordinated fashion, the activation of these kinases. Indeed, we found an accumulation of intracellular cholesterol in melanoma cells (with BRAFV600E mutations) and non-small cell lung cancer cells (with KRASG12C mutations) treated with MAPK and KRASG12C inhibitors, respectively. Importantly, depletion of cholesterol not only prevents the feedback activation of pTyr signaling but also enhances the cytotoxic effects of MAPK pathway inhibitors, both in vitro and in vivo. Together, our findings suggest that cholesterol contributes to the tumor adaptive response upon targeted MAPK pathway inhibitors. These results also suggest that MAPK pathway inhibitors could be combined with cholesterol-lowering agents to achieve a more complete and durable response in tumors with hyperactive MAPK signaling.

Antibiotic Resistance and Epigenetics: More to It than Meets the Eye.

The discovery of antibiotics in the last century is considered one of the most important achievements in the history of medicine. Antibiotic usage has significantly reduced morbidity and mortality associated with bacterial infections. However, inappropriate use of antibiotics has led to emergence of antibiotic resistance at an alarming rate. Antibiotic resistance is regarded as a major health care challenge of this century. Despite extensive research, well-documented biochemical mechanisms and genetic changes fail to fully explain mechanisms underlying antibiotic resistance. Several recent reports suggest a key role for epigenetics in the development of antibiotic resistance in bacteria. The intrinsic heterogeneity as well as transient nature of epigenetic inheritance provides a plausible backdrop for high-paced emergence of drug resistance in bacteria. The methylation of adenines and cytosines can influence mutation rates in bacterial genomes, thus modulating antibiotic susceptibility. In this review, we discuss a plethora of recently discovered epigenetic mechanisms and their emerging roles in antibiotic resistance. We also highlight specific epigenetic mechanisms that merit further investigation for their role in antibiotic resistance.

Single-cell analysis resolves the cell state transition and signaling dynamics associated with melanoma drug-induced resistance.

Continuous BRAF inhibition of BRAF mutant melanomas triggers a series of cell state changes that lead to therapy resistance and escape from immune control before establishing acquired resistance genetically. We used genome-wide transcriptomics and single-cell phenotyping to explore the response kinetics to BRAF inhibition for a panel of patient-derived BRAFV600 -mutant melanoma cell lines. A subset of plastic cell lines, which followed a trajectory covering multiple known cell state transitions, provided models for more detailed biophysical investigations. Markov modeling revealed that the cell state transitions were reversible and mediated by both Lamarckian induction and nongenetic Darwinian selection of drug-tolerant states. Single-cell functional proteomics revealed activation of certain signaling networks shortly after BRAF inhibition, and before the appearance of drug-resistant phenotypes. Drug targeting those networks, in combination with BRAF inhibition, halted the adaptive transition and led to prolonged growth inhibition in multiple patient-derived cell lines.

Environment Shapes the Accessible Daptomycin Resistance Mechanisms in Enterococcus faecium.

Daptomycin binds to bacterial cell membranes and disrupts essential cell envelope processes, leading to cell death. Bacteria respond to daptomycin by altering their cell envelopes to either decrease antibiotic binding to the membrane or by diverting binding away from septal targets. In Enterococcus faecalis, daptomycin resistance is typically coordinated by the three-component cell envelope stress response system, LiaFSR. Here, studying a clinical strain of multidrug-resistant Enterococcus faecium containing alleles associated with activation of the LiaFSR signaling pathway, we found that specific environments selected for different evolutionary trajectories, leading to high-level daptomycin resistance. Planktonic environments favored pathways that increased cell surface charge via yvcRS upregulation of dltABCD and mprF, causing a reduction in daptomycin binding. Alternatively, environments favoring complex structured communities, including biofilms, evolved both diversion and repulsion strategies via divIVA and oatA mutations, respectively. Both environments subsequently converged on cardiolipin synthase (cls) mutations, suggesting the importance of membrane modification across strategies. Our findings indicate that E. faecium can evolve diverse evolutionary trajectories to daptomycin resistance that are shaped by the environment to produce a combination of resistance strategies. The accessibility of multiple and different biochemical pathways simultaneously suggests that the outcome of daptomycin exposure results in a polymorphic population of resistant phenotypes, making E. faecium a recalcitrant nosocomial pathogen.

Drug-induced expression of the RNA-binding protein HuR attenuates the adaptive response to BRAF inhibition in melanoma.

Strategies that aim to limit the adaptive response to pathway inhibition in BRAF-mutated melanoma face the inherent limit of signaling redundancy and multiplicity of possible bypass mechanisms. Drug-induced expression of selected RNA-binding proteins, like the ubiquitously expressed HuR, has the potential to differentially stabilize the expression of many genes involved in the compensatory mechanisms of adaptive response. Here, we detect in BRAF-mutated melanoma cell lines having a higher propensity for adaptive response and in non-responding melanoma tumors, a larger proportion of HuRLow cells in the expression distribution of HuR. Using knockdown experiments, we demonstrate, through expression profiling and phenotypic assays, that increasing the proportion of HuRLow cells favors the adaptive response to BRAF inhibition, provided that the HuRLow state stays reversible. The MAPK dependency of melanoma cells appears to be diminished as the proportion of HuRLow cells increases. In single-cell assays, we demonstrate that the HuRLow cells display plasticity in their growth expression profile. Importantly, the adaptive over-proliferating cells emerge in the subpopulation containing the HuRLow cells. Therapeutic concentrations of lithium salts, although they moderately increase the global expression of HuR, are sufficient to suppress the HuRLow cells, induce an overall less resistant expression profile and attenuate in a HuR-dependent manner the adaptive response of melanoma cells in ex vivo assays. The therapeutic effectiveness of this approach is also demonstrated in vivo in mice xenografts. This study has immediate clinical relevance for melanoma therapy and opens a new avenue of strategies to prevent the adaptive response to targeted cancer therapy.

Loss of Function in Escherichia coli Exposed to Environmentally Relevant Concentrations of Benzalkonium Chloride.

Assessing the risk of resistance associated with biocide exposure commonly involves exposing microorganisms to biocides at concentrations close to the MIC. With the aim of representing exposure to environmental biocide residues, Escherichia coli MG1655 was grown for 20 passages in the presence or absence of benzalkonium chloride (BAC) at 100 ng/liter and 1,000 ng/liter (0.0002% and 0.002% of the MIC, respectively). BAC susceptibility, planktonic growth rates, motility, and biofilm formation were assessed, and differentially expressed genes were determined via transcriptome sequencing. Planktonic growth rate and biofilm formation were significantly reduced (P < 0.001) following BAC adaptation, while BAC minimum bactericidal concentration increased 2-fold. Transcriptomic analysis identified 289 upregulated and 391 downregulated genes after long-term BAC adaptation compared with the respective control organism passaged in BAC-free medium. When the BAC-adapted bacterium was grown in BAC-free medium, 1,052 genes were upregulated and 753 were downregulated. Repeated passage solely in biocide-free medium resulted in 460 upregulated and 476 downregulated genes compared with unexposed bacteria. Long-term exposure to environmentally relevant BAC concentrations increased the expression of genes associated with efflux and reduced the expression of genes associated with outer-membrane porins, motility, and chemotaxis. This was manifested phenotypically through the loss of function (motility). Repeated passage in a BAC-free environment resulted in the upregulation of multiple respiration-associated genes, which was reflected by increased growth rate. In summary, repeated exposure of E. coli to BAC residues resulted in significant alterations in global gene expression that were associated with minor decreases in biocide susceptibility, reductions in growth rate and biofilm formation, and loss of motility.IMPORTANCE Exposure to very low concentrations of biocides in the environment is a poorly understood risk factor for antimicrobial resistance. Repeated exposure to trace levels of the biocide benzalkonium chloride (BAC) resulted in loss of function (motility) and a general reduction in bacterial fitness but relatively minor decreases in susceptibility. These changes were accompanied by widespread changes in the Escherichia coli transcriptome. These results demonstrate the importance of including phenotypic characterization in studies designed to assess the risks of biocide exposure.

Using Reverse Phase Protein Array (RPPA) to Identify and Target Adaptive Resistance.

Tumor cells and the tumor ecosystem rapidly evolve in response to therapy. This tumor evolution results in the rapid emergence of drug resistance that limits the magnitude and duration of response to therapy including chemotherapy, targeted therapy, and immunotherapy. Thus, there is an urgent need to understand and interdict tumor evolution to improve patient benefit to therapy. Reverse phase protein array (RPPA) provides a powerful tool to evaluate and develop approaches to target the processes underlying one form of tumor evolution: adaptive evolution. Tumor cells and the tumor microenvironment rapidly evolve through rewiring of protein networks to bypass the effects of therapy. In this review, we present the concepts underlying adaptive resistance and use of RPPA in understanding resistance mechanisms and identification of effective drug combinations. We further demonstrate that this novel information is resulting in biomarker-driven trials aimed at targeting adaptive resistance and improving patient outcomes.

SOX2-mediated upregulation of CD24 promotes adaptive resistance toward targeted therapy in melanoma.

Melanoma is often characterized by a constitutively active RAS-RAF-MEK-ERK pathway. For targeted therapy, BRAF inhibitors are available that are powerful in the beginning but resistance occurs rather fast. A better understanding of the mechanisms of resistance is urgently needed to increase the success of the treatment. Here, we observed that SOX2 and CD24 are upregulated upon BRAF inhibitor treatment. A similar upregulation was seen in targeted therapy-resistant, melanoma-derived induced pluripotent cancer cells (iPCCs). SOX2 and CD24 are known to promote an undifferentiated and cancer stem cell-like phenotype associated with resistance. We, therefore, elucidated the role of SOX2 and CD24 in targeted therapy resistance in more detail. We found that the upregulation of SOX2 and CD24 required activation of STAT3 and that SOX2 induced the expression of CD24 by binding to its promoter. We find that the overexpression of SOX2 or CD24 significantly increases the resistance toward BRAF inhibitors, while SOX2 knock-down rendered cells more sensitivity toward treatment. The overexpression of CD24 or SOX2 induced Src and STAT3 activity. Importantly, by either CD24 knock-down or Src/STAT3 inhibition in resistant SOX2-overexpressing cells, the sensitivity toward BRAF inhibitors was re-established. Hence, we suggest a novel mechanism of adaptive resistance whereby BRAF inhibition is circumvented via the activation of STAT3, SOX2 and CD24. Thus, to prevent adaptive resistance, it might be beneficial to combine Src/STAT3 inhibitors together with MAPK pathway inhibitors.

Broad-Spectrum Adaptive Antibiotic Resistance Associated with Pseudomonas aeruginosa Mucin-Dependent Surfing Motility.

Surfing motility is a novel form of surface adaptation exhibited by the nosocomial pathogen Pseudomonas aeruginosa in the presence of the glycoprotein mucin, which is found in high abundance at mucosal surfaces, especially those of the lungs of cystic fibrosis and bronchiectasis patients. Here, we investigated the adaptive antibiotic resistance of P. aeruginosa under conditions in which surfing occurs compared that in to cells undergoing swimming. P. aeruginosa surfing cells were significantly more resistant to several classes of antibiotics, including aminoglycosides, carbapenems, polymyxins, and fluoroquinolones. This was confirmed by incorporation of antibiotics into growth medium, which revealed a concentration-dependent inhibition of surfing motility that occurred at concentrations much higher than those needed to inhibit swimming. To investigate the basis of resistance, transcriptome sequencing (RNA-Seq) was performed and revealed that surfing influenced the expression of numerous genes. Included among genes dysregulated under surfing conditions were multiple genes from the Pseudomonas resistome; these genes are known to affect antibiotic resistance when mutated. Screening transposon mutants in these surfing-dysregulated resistome genes revealed that several of these mutants exhibited changes in susceptibility to one or more antibiotics under surfing conditions, consistent with a contribution to the observed adaptive resistance. In particular, several mutants in resistome genes, including armR, recG, atpB, clpS, nuoB, and certain hypothetical genes, such as PA5130, PA3576, and PA4292, showed contributions to broad-spectrum resistance under surfing conditions and could be complemented by their respective cloned genes. Therefore, we propose that surfing adaption led to extensive multidrug adaptive resistance as a result of the collective dysregulation of diverse genes.