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Growth Inhibitory effect of cisplatin.

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Primers and shIDO sequences.

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Expression levels of TPH, IDO2 and TDO2.

Increased OXPHOS was found in CR cells.

Relative mRNA levels of CYP1B1.

Immune check point inhibitor(s) have become a main stay of treatment for melanoma regardless of its mutational status. However, not all patients respond to treatment. In fact, only 40-60% respond to treatment. Currently, only PD-L1/PD-1 expression and mutational burden can predict response to treatment. Published data have shown that exosomal PD-L1 is an important factor contributing to response/failure to checkpoint inhibitor treatment. Exosome is known to carry several types of cargoes and is one of the important delivery systems in the body. We have begun to examine the role of exosomal PD-L1, PD-1 in predicting response, and as well as how to overcome drug resistance. We initiated an institutional study in metastatic melanoma patients who underwent immunotherapy treatment (anti-PD-1 + anti-CTLA 4 drugs). Blood samples were obtained at baseline and at subsequent treatment. Both serum and plasma exosomes were isolated. Eighteen patients were entered in the study. We have found that 1) Patients with PD-L1 negative tumor can have high exosomal PD-L1 expression, 2) all patients had exosomal PD-L1, and 3) all patients with decreased exosomal PD-L1 expression after treatment showed antitumor response [partial response (PR) or complete response (CR)]. In contrast, patients who had consistently high exosomal PD-L1 expression post-treatment showed no response, and this can be seen after the third cycle of treatment. Patients who achieved CR showed low exosomal PD-L1 and no exosomal PD-1 post-treatment. Importantly, we have also found that exosomes carry antiapoptotic protein and proapoptotic protein, as well as ATG family protein, which is related to autophagy. Whether these proteins also play a role in determining the cell fate in the metastatic sites is not known and will need future investigation. Interestingly, the two patients who showed no response also had high p-AKT. Since traditional anti PD-1 and anti-PD-L1 drugs do not have effect on exosomal PD-L1 and PD-1, we formed a collaboration with Dr. Stephane Roche at Florida Atlantic University, who has designed novel peptides by exploiting the innate plasticity of the PD-1 receptor (pembrolizumab H3 loop mimics). Thus far, we have screened many compounds and have selected 2 compounds for further testing. Together, we have also designed new PD-L1 peptides that can inhibit both PD-L1 and PI3K/AKT pathways simultaneously. Hence one can overcome checkpoint inhibitor resistance due to exosomal PD-L1/PD-1. Supported by grant from Sylvester Comprehensive Cancer Center and R21GM132754. Citation Format: Pablo Eduardo Puente, Dan Nguyen, Niramol Savaraj, Chunjing Wu, Medhi Wangpaichitr, Stephane Roche, Guangkuan Zhao, Alexis Richaud, Jose Lutzky, Leonel Hernandez Aya, Mecker Moller, Jessica Crystal, Neha Goel, Lynn Feun. Can exosomal cargoes predict checkpoint inhibitor response in melanoma, and can we overcome drug resistance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4114.

Cisplatin resistance is a major barrier in the effective treatment of lung cancer. Cisplatin-resistant (CR) lung cancer cells do not primarily use glucose but rather consume amino acids such as glutamine and tryptophan (Trp) for survival. CR cells activate the kynurenine (KYN) pathway (KP) to cope with excessive reactive oxygen species (ROS) and maintain homeostasis for growth and proliferation. Consequently, indoleamine 2,3-dioxygenase-1 (IDO1) becomes an essential enzyme for CR cells' survival because it initiates and regulates the first step in the KP. Increased IDO1 activities and ROS levels are found in CR cells versus cisplatin-sensitive lung cancer. Importantly, significantly greater KYN/Trp ratio (P = 0.005) is detected in serum of patients who fail cisplatin when compared with naïve treatment. Knocking down IDO1 using shRNA or IDO1 inhibitors heightens ROS levels and results in a significant growth inhibitory effect only on CR cells and not on cisplatin-sensitive cells. Exposing CR cells to antioxidant (TIRON) results in suppression of IDO1 activity and confers resistance to IDO1 inhibition, indicating an interrelationship between ROS and IDO1. Because KYN plays a critical role in reprogramming naïve T cells to the immune-suppressive regulatory T-cell (T-reg) phenotype, we observed higher expression of TGFβ, FoxP3, and CD4+CD25+ in mice bearing CR tumors compared with tumors from cisplatin-sensitive counterparts. Implications: Findings suggest that the enzyme-inhibitory activity and antitumor efficacy of IDO1 inhibitors rely in part on ROS levels, arguing that IDO1 expression alone may be insufficient to determine the clinical benefits for this class of experimental cancer drugs. Importantly, IDO1 inhibitors may be more suitable to treat patients with lung cancer who failed cisplatin therapy than naïve treatment patients.

The development of drug resistance in tumors is a major obstacle to effective cancer chemotherapy and represents one of the most significant complications to improving long-term patient outcomes. Despite early positive responsiveness to platinum-based chemotherapy, the majority of lung cancer patients develop resistance. The development of a new combination therapy targeting cisplatin-resistant (CR) tumors may mark a major improvement as salvage therapy in these patients. The recent resurgence in research into cellular metabolism has again confirmed that cancer cells utilize aerobic glycolysis (“the Warburg effect”) to produce energy. Hence, this observation still remains a characteristic hallmark of altered metabolism in certain cancer cells. However, recent evidence promotes another concept wherein some tumors that acquire resistance to cisplatin undergo further metabolic alterations that increase tumor reliance on oxidative metabolism (OXMET) instead of glycolysis. Our review focuses on molecular changes that occur in tumors due to the relationship between metabolic demands and the importance of NAD+ in redox (ROS) metabolism and the crosstalk between PARP-1 (Poly (ADP ribose) polymerase-1) and SIRTs (sirtuins) in CR tumors. Finally, we discuss a role for the tumor metabolites of the kynurenine pathway (tryptophan catabolism) as effectors of immune cells in the tumor microenvironment during acquisition of resistance in CR cells. Understanding these concepts will form the basis for future targeting of CR cells by exploiting redox-metabolic changes and their consequences on immune cells in the tumor microenvironment as a new approach to improve overall therapeutic outcomes and survival in patients who fail cisplatin.

We discovered a crucial change common to all platinum resistant (CR) NSCLC cells, wherein a high level of reactive oxygen species (ROS) modulates tumor metabolism. CR cells activate the kynurenine (KYN) pathway (KP) during excessive oxidative stress to maintain high ROS for proliferation by catabolizing tryptophan (TRP) to KYN via indoleamine 2,3-dioxygenase-1 (IDO1); and by expressing (3-5x) higher levels of programmed death-ligand 1 (PD-L1) compared to platinum sensitive cells (p Citation Format: Sydney Spector, Chunjing Wu, Dan Nguyen, George Theodore, Ashley Garcia, Lynn Feun, Niramol Savaraj, Medhi Wangpaichitr. Targeting kynurenine pathway using novel IDO/TDO dual inhibitor (AT0174) to modulate tumor microenvironment in platinum resistant non-small cell lung cancer cancer: An immunometabolism compliment markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2325.

Background: Hepatocellular carcinoma (HCC) remains one of the most common cancers worldwide. Immunotherapy with various check point inhibitors has been shown to have antitumor activity in HCC. The combination of Atezolizumab plus Bevacizumab resulted in better overall survival and progression-free survival outcome than sorafenib and is now approved for first line for the treatment of HCC. Other PD-1 inhibitors, nivolumab and pembrolizumab, have been shown to have antitumor activity with an overall response rate 20-30% as second line agents. The important question is whether one can find biomarkers which could predict response. Methods: Plasma biomarkers were measured by ELISA before and during treatment with pembrolizumab for advanced unresectable HCC patients. MiR RNA was carried out by Ocean Ridge Biosciences. Circulating MDSC (CD14+ CD33+ HLA-DR (low) Treg CD4+ CD25+NK cells and exhausted CD8 re-invigoration (Ki 67 in PD-1 + CD8+) were analyzed by flow cytometry. Results: Serum IL-1β, Il-6, IL-12, IL18, IFN-γ, IL-10, CXCL9, CCL4, CCl5, and circulating PD-L1 were not predictive of response. However, baseline serum TGF-β is one of the predictors for lack of response. Using a cutoff point of 200 pg/ml, TGF-β is also a predictor for progression free survival (P=0.008) and overall survival (p=0.005). In addition, several candidate miRNA were detected higher in responders compared to non-responders. Among them, three miRNAs:1) HsamiR-145b-3p which functions to suppress CD4+ T cell differentiation into Th2/Th17, a tumor suppressor targeting oncogene (i.e. DNMT3 b), 2 ) Has-miR93-5p which targets IL-8, VEGF, diminishing CXCL12 and PD-L1 expression and 3) Has-miR-23b-3b which regulates inflammatory factors (including IL-6, TNF alpha VCAM-1) are consistently higher in the responders compared to non-responders. Among immune cells analyzed, only MDSC (CD14+ CD33+ HLA-DR low) was a good predictor for responders. The responders had a lower MDSC (4.2) and non-responders had high (56.2, p=0.047). Using a cutoff point of 25, MDSC was also a predictor for overall survival (p= 0.026). Both circulating Treg and exhausted CD8 re -invigoration exhibited no differences between responders and non-responders. (12.8 vs 6.2 for Treg and 18.7 vs 25.7 for PD1+ CD8+.T cell re-invigoration, respectively). Conclusion: Serum TGF-β, MDSC, and miR -145b-3p, miR94-5p, miR -23b-3p are possible circulating biomarkers which one can use to design future clinical trials. In addition, combination therapy with TGF-β inhibitor and checkpoint inhibitor is possible to improve response and survival in these HCC patients, and should be evaluated in a prospective clinical trial. Supported in part by a grant from Sylvester Comprehensive Cancer Center. Citation Format: Lynn G. Feun, Ying-Ying Li, Chunjing Wu, Medhi Wangpaichitr, Alfred Chicco, Niramol Savaraj. Circulating biomarkers to predict antitumor response to immunotherapy in advanced unresectable hepatoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2771.

The coefficients of thermal expansion (CTE) and thermal conductivity (TC) are important for heat sink applications, as they can minimize stress between heat sink substrates and chips and prevent failure from thermal accumulation in electronics. We investigated the interface behavior and manufacturing of diamond/Cu composites and found that they have much lower TCs than copper due to their low densities. Most defects, such as cavities, form around diamond particles, substantially decreasing the high TC of diamond reinforcements. However, the measurement results for the Cu-coated diamond/Cu composites are unsatisfactory because the nanosized copper layer on the diamond surface grew and spheroidized at elevated sintering temperatures. Realizing ideal interfacial bonding between a copper matrix and diamond particles is difficult. The TC of the 40 vol.% Ti-coated diamond/Cu composite is 475.01 W m&minus, 1 K&minus, 1, much higher than that of diamond/Cu and Cu-coated diamond/Cu composites under equivalent manufacturing conditions. The minimally grown titanium layer retained its nanosized and was consistent with the sintering temperature. Depositing a nanosized titanium layer on a diamond surface will strengthen interfacial bonding through interface reactions among the copper matrix, nanosized titanium layer and diamond particles, reducing the interfacial thermal resistance and exploiting the high TC of diamond particles, even if defects from powder metallurgy remain. These results provide an important experimental and theoretical basis for manufacturing diamond/Cu composites for heat sink applications.

A liquid-solid separation technology (LSS) is applied to fabricate near-net shape packaging shells made of hypereutectic Al-Si composites with gradient distributions of the microstructure and properties. This new technology solves the problem of the formation of coarse flake primary Si, which will seriously deteriorate the mechanical properties of the materials, and unavoidable with conventional casting method. The microstructure of the hypereutectic Al-Si composite were characterized using scanning electron microscope, energy dispersive spectroscopy and transmission electron microscope. In order to eliminate the influence of crystal defects and stress, the effects of annealing treatment on the thermal expansion coefficient and thermal conductivity of the composites were also investigated. The results show that the volume fraction of Si decreases along a gradient from the bottom centre of the packaging shell, which houses the chips, to the top of the packaging wall, which is welded with the cover materials. The bottom of the shell has a CTE of 11.7 × 10−6 K−1, TC of 124.3 W m-1 K-1, and primary Si content of 53.7%. The top of the shell has a CTE of 18.4 × 10−6 K−1, TC of 151.2 W m-1 K-1, and primary Si content of 23.3%. Such a distribution enables a good matching of the thermal expansion behaviour of the packaging shell with the chips, which have typically low CTE, while avoiding the issue of poor soldering performance of Al/Si alloys. In addition, it reduces the internal stress of the shell due to integrated forming technology.

Melanomas harboring BRAF mutation (V600E) are known to recur frequently following treatment with BRAF inhibitors (BRAFi) despite a high initial response rate. Our previous study has uncovered that BRAFi-resistant melanoma (BR) cells are vulnerable to arginine deprivation. It has been reported that naive melanoma cells undergo autophagy and re-express argininosuccinate synthetase 1 (ASS1) to enable them to synthesize arginine for survival when encountering arginine deprivation. Abolishing these two factors in BR cells confers sensitivity to arginine deprivation. In this report, we further demonstrated that downregulation of AMPK-α1 in BR cells is a major factor contributing to impairment of autophagy as evidenced by decreased autophagosome formation. These BR cells also showed a metabolic shift from glucose to arginine dependence, which was supported by decreased expressions of GLUT1 (glucose transporter) and hexokinase II (HKII) coupled with less glucose uptake but high levels of arginine transporter CAT-2 expression. Furthermore, silencing CAT-2 expression also distinctly attenuated BR cell proliferation. Notably, when naive melanoma cells became BR cells by long-term exposure to BRAFi, a stepwise degradation of AMPK-α1 was initiated via ubiquitin-proteasome system (UPS). We discovered that a novel E3 ligase, RING finger 44 (RNF44), is responsible for promoting AMPK-α1 degradation in BR cells. RNF44 expression in BR cells was upregulated by transcription factor CREB triggered by hyperactivation of ERK/AKT. High levels of RNF44 corresponding to low levels of AMPK-α1 appeared in BR xenografts and melanoma tumor samples from BR and BRAFi/MEK inhibitor (MEKi)-resistant (BMR) melanoma patients. Similar to BR cells, BMR cells were also sensitive to arginine deprivation. Our study provides a novel insight into the mechanism whereby BRAFi or BRAFi/MEKi resistance drives proteasomal degradation of AMPK-α1 and consequently regulates autophagy and metabolic reprogramming in melanoma cells.

Apoptotic and immunological cell death are the two major pathways that can lead to demise of cancer cells. Targeting both pathways by combining chemotherapy and checkpoint inhibitors has proven to be more effective in treating lung cancer but not in melanoma, due to the fact that melanoma cells often express high levels of antiapoptotic proteins Bcl-2 ( 60%) which contribute to their resistance to chemotherapy. We have previously shown that melanoma cell lines with high Bcl-2 expression , regardless of their BRAF mutation, do not possess high PD-L1 ( AACR abstract #4747 2019). This is also seen in melanoma tissue obtained from patients. Here, we attempt to explore whether there is any relationship between Bcl-2 and PD-L1 in melanoma cells using knock in and knock out methods. Two melanoma cells derived from patients ( mel1220 which possess high levels of Bcl-2 and Mel-F which possess high levels of PD-L1 ) and their BRAF/MEK counterparts, Mel-1220 BMR and MelF BMR, respectively, were used for the study. Interestingly, cell lines with high Bcl-2 , their BMR counterparts showed no changes in Bcl-2 levels, However, in cell lines with high PD-L1 expression, the levels of PD-L1 is even higher when they become BMR. This could explain why after failure to BRAF/MEK inhibitor , the clinical response to anti PD-1 check point inhibitors varies. Next, we transfected PD-L1 using the SP-GFP-CD274-NU Vector ( purchased from Addgene )into the BcL-2 over expressing cell lines. Three clones were selected. Two clones ( 1220.PD-L1/1 and 1220 PD-L1/3) showed only slight decreased Bcl-2 expression ( less than 1 fold) and in one clone ( 1220.PD-L1/2), there is no effect seen. Furthermore, there is no discernable change in other antiapoptotic proteins ( Bcl-xl, BAX, Mcl-2 survivin, NOXA and PUMA ) by westernblot noted. Similar results were obtained with their BMR counterparts. Total knock down of Bcl-2 using CRISPR knockout ( kit purchased from Origene) results in cell death. In contrast, transfection of Bcl2 into PD-L1 overexpression results in decreased PD-L1 expression by 2-4 fold both by PCR and by westernblot in both clones. however , there is no discernable changes on surface PD-L1 by flow cytometery. Transfection of BCl-2 also does not affect other BCL-2 family proteins. Similar results were obtained with their BMR counterparts. From these data, it appears that the presence of Bcl-2 ( RNA/protein) could affect intrinsic PD-L1 expression, however, the surface PD-L1 which is the key to bind PD-1 did not change. On the other hand, the presence of PD-L1 RNA/protein has no discernable effect on BcL-2 expression. Whether partial knock down BCl-2 or treatment with Bcl-2 inhibitor can lead to increased PD-L1 expression is not known and is under investigation. Overall, it appears that combination of Bcl-2 inhibitor and check point inhibitor may be more effective in melanoma patients with high Bcl-2 expression. (Supported by VA merit review award; 1BX003328) Citation Format: Niramol Savaraj, Min You, Ying-Ying Li, Chunjing Wu, Medhi Wangpaichitr, Saurez Miguel, Alfred Chicco, Macus T. Kuo, Lynn G. Feun. Targeting immunological and apoptotic cell death to improve therapeutic efficacy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 988.

Arginine (Arg) deprivation can inhibit tumors which do not express argininosuccinate synthetase (ASS), a key enzyme for synthesis of Arg. Arg deiminase (ADIPEG20) has shown antitumor activity in such tumors, including melanoma and mesothelioma. Resistance to this treatment occurs due to re-expression of ASS. We have shown that this is mediated via cMyc (positive regulator) and HIF1α ( negative regulator ). Arg starvation rapidly induces chromatin remodeling complex P300-HDAC2-Sin3A which epigenetically deacetylates H3K14ac and H3K27ac at the ASS promoter. Following the PHD2-derived HIF1α-degrading system, the promoter-bound HIF1α is degraded (Sci Rep 7:10814). This allows cMyc (an E-Box binder), to turn on ASS. Arg starvation rapidly triggers externalization of Gas6 to interact with its receptor tyrosine kinase (RTK) Axl. This activates the downstream Ras-PI3K/AKT/GSK3β pathway, resulting in stabilization of cMyc. However, this activation also turns on PDL1 gene expression. This is not surprising since cMyc has been shown to bind to PDL1 promoter and increase its expression. Thus, re-expression of ASS also leads to PDL1 expression. We studied these findings in two other cell lines, Mel114 and Skmel2, and obtained similar results, Interestingly, in three cell lines with high levels of antiapoptotic protein Bcl-2, Arg starvation only leads to growth inhibition, with only less than 2 fold increase in ASS and no PDL1 expression. These results suggest that increased Bcl-2 expression negates the necessity to increase PDL1 and ASS to prevent cell death from nutritional stress. In contrast, transfection of ASS in A2058, A375, Mel1220 and SkMel2 will only confer resistance to Arg starvation, and does not affect PDL1, Bcl2 expression or other alterations in PI3K/AKT pathway. Furthermore, in samples from patients who failed ADIPEG20 due to increased ASS expression, the primary cultures or re-biopsy tumor also exhibited high levels of PDL1 expression, which most likely will make them susceptible to anti PDL1 treatment. On the other hand, six primary cultures derived from patients who failed BRAF/MEK inhibitor (BMR) also showed low levels of ASS and increase in PDL1 expression (1.5-4 fold increase both in mRNA and proteins level compared to their parental cells). These results also applied to 5 pairs of BMR cell lines (A375/BMR, A2058/BMR, Skmel2/BMR, UACC62/BMR, MelRR/BMR). These cells also are very susceptible to combination of ADIPEG20 and PD-L1 treatment. Overall, our data suggest that combination of Arg deprivation and check point inhibitors is effective in patients whose tumors do not express ASS and as salvage therapy for BRAF/MEK inhibitor resistant patients. Supported by the VA Merit Review Award(1BX003328) Citation Format: Lynn G. Feun, Chunjing Wu, Ying-Ying Li, Min You, Medhi Wangpaichitr, Miguel Suarez, Niramol G. Savaraj. Eradicating melanoma cells using combination of nutritional stress and checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2336.

Cisplatin resistance remains a major problem in the treatment of lung cancer. We have discovered that cisplatin resistant (CR) lung cancer cells, regardless of the signaling pathway status, share the common parameter which is an increase in reactive oxygen species (ROS) and undergo metabolic reprogramming. CR cells were no longer addicted to the glycolytic pathway, but rather relied on oxidative metabolism. They took up twice as much glutamine and were highly sensitive to glutamine deprivation. Glutamine is hydrolyzed to glutamate for glutathione synthesis, an essential factor to abrogate high ROS via xCT antiporter. Thus, blocking glutamate flux using riluzole (an amyotropic lateral sclerosis approved drug) can selectively kill CR cells in vitro and in vivo. However, we discovered here that glutathione suppression is not the primary pathway in eradicating the CR cells. Riluzole can lead to further decrease in NAD(+) (nicotinamide adenine dinucleotide) and lactate dehydrogenase-A (LDHA) expressions which in turn further heightened oxidative stress in CR cells. LDHA knocked-down cells became hypersensitive to riluzole treatments and possessed increased levels of ROS. Addition of NAD(+) re-stabilized LDHA and reversed riluzole induced cell death. Thus far, no drugs are available which could overcome cisplatin resistance or kill cisplatin resistant cells. CR cells possess high levels of ROS and undergo metabolic reprogramming. These metabolic adaptations can be exploited and targeted by riluzole. Riluzole may serve as a dual-targeting agent by suppression LDHA and blocking xCT antiporter. Repurposing of riluzole should be considered for future treatment of cisplatin resistant lung cancer patients., Department of Veterans Affairs, BLR&D Career Development Award-2; NIH/NCI; Woman Cancer Association of the University of Miami Award

Background Checkpoint inhibitors have shown modest activity in patients with advanced hepatocellular carcinoma (HCC). Herein, the authors report a prospective single-institution clinical/translational phase 2 study of pembrolizumab in patients with advanced HCC and circulating biomarkers closely related to response. Methods Pembrolizumab was administered at a dose of 200 mg intravenously every 3 weeks among patients who may have developed disease progression while receiving, were intolerant of, or refused sorafenib. The circulating levels of cytokines, chemokines, programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), and PD-L2 were correlated with response, tumor PD-L1 expression, and other clinicopathological features. Results A total of 29 patients were treated and 28 patients were evaluable for response. The most common laboratory grade 3/4 adverse events were increases in aspartate aminotransferase and/or alanine aminotransferase and serum bilirubin, which for the most part were reversible. In terms of efficacy, one patient achieved a complete response and 8 patients achieved partial responses for an overall response rate of 32%. Four other patients had stable disease. The median progression-free survival was 4.5 months and the median overall survival was 13 months. Response did not correlate with prior sorafenib therapy, PD-L1 tumor staining, or a prior history of hepatitis. Correlative studies revealed that high baseline plasma TGF-β levels (≥200 pg/mL) significantly correlated with poor treatment outcomes after pembrolizumab. Tumor PD-L1 and plasma PD-L1/PD-1 levels were associated with plasma IFN-γ or IL-10. Conclusions Pembrolizumab was found to demonstrate activity in patients with advanced HCC. Toxicity generally was tolerable and reversible. A set of immunological markers in blood plasma as well as PD-L1 staining indicated that baseline TGF-β could be a predictive biomarker for response to pembrolizumab.

Low-cost and good quality Al–diamond composites were prepared in the application of heat dissipation using an advanced liquid–solid separation technology. Their thermal conductivity (TC) and coefficient of thermal expansion (CTE) were measured, especially on the effect of diamond particle surface metallization. The TC of composites reinforced with Ni-coated diamond particles was found to have increased by almost 80 % compared to that of Al alloys. The diamond surface in the composites was characterized using an electron microprobe, X-ray diffraction and SEM. It was found that high TC of the composites was obtained due to the generation of Al4Ni3 and AlNi phases at their interfaces between Al and diamond. In addition, the theoretical calculation for TC and CTE of the composites show the similar tendency with their experimental data.

BRAF inhibitor (BRAFi) has been used for treatment of melanomas harboring V600E mutation. Despite a high initial response rate, resistance to BRAFi is inevitable. Here, we demonstrate that BRAFi-resistant (BR) melanomas are susceptible to arginine deprivation due to inability to initiate re-expression of argininosuccinate synthetase (ASS1, a key enzyme for arginine synthesis) as well as ineffective autophagy. Autophagy and ASS1 re-expression are known to protect melanoma cells from cell death upon arginine deprivation. When melanoma cells become BR cells by long-term in vitro incubation with BRAFi, c-Myc-mediated ASS1 re-expression and the levels of autophagy-associated proteins (AMPK-α1 and Atg5) are attenuated. Furthermore, our study uncovers that downregulation of deubiquitinase USP28 which results in more active c-Myc degradation via ubiquitin-proteasome machinery is the primary mechanism for inability to re-express ASS1 upon arginine deprivation in BR cells. Overexpression of USP28 in BR cells enhances c-Myc expression and hence increases ASS1 transcription upon arginine deprivation, and consequently leads to cell survival. On the other hand, overexpression of Atg5 or AMPK-α1 in BR cells can redirect arginine deprivation-induced apoptosis toward autophagy. The xenograft models also confirm that BR tumors possess lower expression of ASS1 and are hypersensitive to arginine deprivation. These biochemical changes in BRAFi resistance which make them vulnerable to arginine deprivation can be exploited for the future treatment of BR melanoma patients.

Loss of argininosuccinate synthetase (ASS) expression in melanoma makes these tumor cells vulnerable to arginine deprivation. Pegylated arginine deiminase (ADI-PEG20) which degrades arginine to citrulline and ammonia has been used clinically and partial responses and stable disease have been noted with minimal toxicity. In order to improve the therapeutic efficacy of ADI-PEG20, we have combined ADI-PEG20 with a DNA damaging agent, cisplatin. We have shown that the combination of the two drugs together significantly improved the therapeutic efficacy when compared to ADI-PEG20 alone or cisplatin alone in 4 melanoma cell lines, regardless of their BRAF mutation. In-vivo study also exhibited the same effect as in-vitro with no added toxicity to either agent alone. The underlying mechanism is complex, but increased DNA damage upon arginine deprivation due to decreased DNA repair proteins, FANCD2, ATM, and CHK1/2 most likely leads to increased apoptosis. This action is further intensified by increased proapoptotic protein, NOXA, and decreased antiapoptotic proteins, SURVIVIN, BCL2 and XIAP. The autophagic process which protects cells from apoptosis upon ADI-PEG20 treatment also dampens upon cisplatin administration. Thus, the combination of arginine deprivation and cisplatin function in concert to kill tumor cells which do not express ASS without added toxicity to normal cells.