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Purpose: PAX-fusion negative rhabdomyosarcoma (FN RMS) is driven by alterations in the RAS/MAP kinase pathway and is partially responsive to MEK inhibition. Overexpression of IGF1R and its ligands is also observed in FN RMS. Preclinical and clinical studies have suggested that IGF1R is itself an important target in FN RMS. Our previous studies revealed preclinical efficacy of the MEK1/2 inhibitor, trametinib, and an IGF1R inhibitor, BMS-754807, but this combination was not pursued clinically due to intolerability in preclinical murine models. Here, we sought to identify a combination of an MEK1/2 inhibitor and IGF1R inhibitor, which would be tolerated in murine models and effective in both cell line and patient-derived xenograft models of RAS-mutant FN RMS. Experimental Design: Using proliferation and apoptosis assays, we studied the factorial effects of trametinib and ganitumab (AMG 479), a mAb with specificity for human and murine IGF1R, in a panel of RAS-mutant FN RMS cell lines. The molecular mechanism of the observed synergy was determined using conventional and capillary immunoassays. The efficacy and tolerability of trametinib/ganitumab was assessed using a panel of RAS-mutated cell-line and patient-derived RMS xenograft models. Results: Treatment with trametinib and ganitumab resulted in synergistic cellular growth inhibition in all cell lines tested and inhibition of tumor growth in four of six models of RAS-mutant RMS. The combination had little effect on body weight and did not produce thrombocytopenia, neutropenia, or hyperinsulinemia in tumor-bearing SCID beige mice. Mechanistically, ganitumab treatment prevented the phosphorylation of AKT induced by MEK inhibition alone. Therapeutic response to the combination was observed in models without a mutation in the PI3K/PTEN axis. Conclusions: We demonstrate that combined trametinib and ganitumab is effective in a genomically diverse panel of RAS-mutated FN RMS preclinical models. Our data also show that the trametinib/ganitumab combination likely has a favorable tolerability profile. These data support testing this combination in a phase I/II clinical trial for pediatric patients with relapsed or refractory RAS-mutated FN RMS.

A principal protective component of the mammalian blood-brain barrier (BBB) is the high expression of the multidrug efflux transporters P-glycoprotein (P-gp, encoded byABCB1) and ABCG2 (encoded byABCG2) on the luminal surface of endothelial cells. The zebrafish P-gp homolog Abcb4 is expressed at the BBB and phenocopies P-gp. Comparatively little is known about the four zebrafish homologs of the humanABCG2gene:abcg2a, abcg2b, abcg2c, andabcg2d. Here we report the functional characterization and brain tissue distribution of zebrafish ABCG2 homologs. To determine substrates of the transporters, we stably expressed each in HEK-293 cells and performed cytotoxicity and fluorescent efflux assays with known ABCG2 substrates. We found Abcg2a had the greatest substrate overlap with ABCG2, and Abcg2d appeared to be the least functionally similar. Using RNAscopein situhybridization we identifiedabcg2aas the only homolog expressed at the adult and larval zebrafish BBB, based on its localization to claudin-5 positive brain vasculature. These results demonstrate the conserved function of zebrafish Abcg2a and suggest that zebrafish may be an appropriate model organism for studying the role of ABCG2 at the BBB.

Supplementary Figures 1-7 and Supplementary Table 1

Supplemental tables 1-3, supplemental figure legends, supplemental figures 1-4

Pharmacodynamic response to trametinib/ganitumab in xenograft models does not predict tumor shrinkage

Related to Figure 4

Purpose:PAX-fusion negative rhabdomyosarcoma (FN RMS) is driven by alterations in the RAS/MAP kinase pathway and is partially responsive to MEK inhibition. Overexpression of IGF1R and its ligands is also observed in FN RMS. Preclinical and clinical studies have suggested that IGF1R is itself an important target in FN RMS. Our previous studies revealed preclinical efficacy of the MEK1/2 inhibitor, trametinib, and an IGF1R inhibitor, BMS-754807, but this combination was not pursued clinically due to intolerability in preclinical murine models. Here, we sought to identify a combination of an MEK1/2 inhibitor and IGF1R inhibitor, which would be tolerated in murine models and effective in both cell line and patient-derived xenograft models of RAS-mutant FN RMS.Experimental Design:Using proliferation and apoptosis assays, we studied the factorial effects of trametinib and ganitumab (AMG 479), a mAb with specificity for human and murine IGF1R, in a panel of RAS-mutant FN RMS cell lines. The molecular mechanism of the observed synergy was determined using conventional and capillary immunoassays. The efficacy and tolerability of trametinib/ganitumab was assessed using a panel of RAS-mutated cell-line and patient-derived RMS xenograft models.Results:Treatment with trametinib and ganitumab resulted in synergistic cellular growth inhibition in all cell lines tested and inhibition of tumor growth in four of six models of RAS-mutant RMS. The combination had little effect on body weight and did not produce thrombocytopenia, neutropenia, or hyperinsulinemia in tumor-bearing SCID beige mice. Mechanistically, ganitumab treatment prevented the phosphorylation of AKT induced by MEK inhibition alone. Therapeutic response to the combination was observed in models without a mutation in the PI3K/PTEN axis.Conclusions:We demonstrate that combined trametinib and ganitumab is effective in a genomically diverse panel of RAS-mutated FN RMS preclinical models. Our data also show that the trametinib/ganitumab combination likely has a favorable tolerability profile. These data support testing this combination in a phase I/II clinical trial for pediatric patients with relapsed or refractory RAS-mutated FN RMS.

Supplementary Figures S1-S4 from Evidence for Microtubule Target Engagement in Tumors of Patients Receiving Ixabepilone

Supplementary Data from Escape from hsa-miR-519c enables drug-resistant cells to maintain high expression of ABCG2

Purpose: P-glycoprotein (Pgp) antagonists have been difficult to develop because of complex pharmacokinetic interactions and a failure to show meaningful results. Here we report the results of a pharmacokinetic and pharmacodynamic trial using a third-generation, potent, noncompetitive inhibitor of Pgp, tariquidar (XR9576), in combination with docetaxel.Experimental Design: In the first treatment cycle, the pharmacokinetics of docetaxel (40 mg/m2) were evaluated after day 1 and day 8 doses, which were administered with or without tariquidar (150 mg). 99mTc-sestamibi scanning and CD56+ mononuclear cell rhodamine efflux assays were conducted to assess Pgp inhibition. In subsequent cycles, 75 mg/m2 docetaxel was administered with 150 mg tariquidar every 3 weeks.Results: Forty-eight patients were enrolled onto the trial. Nonhematologic grade 3/4 toxicities in 235 cycles were minimal. Tariquidar inhibited Pgp-mediated rhodamine efflux from CD56+ cells and reduced 99mTc-sestamibi clearance from the liver. There was striking variability in basal sestamibi uptake; a 12% to 24% increase in visible lesions was noted in 8 of 10 patients with lung cancer. No significant difference in docetaxel disposition was observed in pairwise comparison with and without tariquidar. Four partial responses (PR) were seen (4/48); 3 in the non–small cell lung cancer (NSCLC) cohort, measuring 40%, 57%, and 67% by RECIST, and 1 PR in a patient with ovarian cancer.Conclusions: Tariquidar is well tolerated, with less observed systemic pharmacokinetic interaction than previous Pgp antagonists. Variable effects of tariquidar on retention of sestamibi in imageable lung cancers suggest that follow-up studies assessing tumor drug uptake in this patient population would be worthwhile. Clin Cancer Res; 17(3); 569–80. ©2010 AACR.

Purpose: Microtubule-stabilizing agents, such as taxanes, have been shown to be effective anticancer drugs. α-Tubulin, a basic unit of microtubules, can undergo several posttranslational modifications after assembly into stabilized microtubules, including acetylation and detyrosination. These modifications have been observed in cell cultures after exposure to microtubule stabilizers. Our objective was to develop a straightforward and dependable assay to show tubulin target engagement in tumor tissue after treatment of patients with ixabepilone(BMS-247550; Ixempra).Experimental Design: Levels of posttranslationally modified α-tubulin were assessed in lysates of cultured malignant cell lines, as well as in both tumor tissue and peripheral blood mononuclear cells derived from patients before and after treatment with ixabepilone. Modification-specific antibodies permitted quantitative Western blot analysis.Results: In cultured cell lines, the levels of detyrosinated (glu-terminated) and acetylated α-tubulin increased after microtubule stabilization induced by ixabepilone. ixabepilone treatment also induced a 2-fold to 25-fold increase in detyrosinated α-tubulin levels in 11 of 13 serial biopsies and a 2-fold to 100-fold increase in acetylated α-tubulin in 11 of 12 serial biopsies obtained from patients receiving ixabepilone. Overall, little or no difference in tubulin modifications were observed between the before and after ixabepilone treatment in lysates from their peripheral blood mononuclear cells at the time point examined.Conclusion: Assessing the levels of detyrosinated and/or acetylated α-tubulin seems to provide a simple and reliable assay to show target engagement by the microtubule-stabilizing agent ixabepilone. Such analyses may provide further understanding of therapeutic success or failure of microtubule-stabilizing agents in cancer therapy.

Overexpression of ABCG2 has been reported in cell lines selected for drug resistance and it is widely believed to be important in the clinical pharmacology of anticancer drugs. We and others have previously identified and validated two microRNAs (miRNA; hsa-miR-519c and hsa-miR-520h) targeting ABCG2. In this study, the shortening of the ABCG2 3′ untranslated region (3′UTR) was found to be a common phenomenon in several ABCG2-overexpressing resistant cell lines, which as a result removes the hsa-miR-519c binding site and its repressive effects on mRNA stability and translation blockade, thereby contributing to drug resistance. On the other hand, reduced expression of hsa-miR-520h, previously thought to have allowed ABCG2 overexpression, was found to be caused by the sequestering of the miRNA by the highly expressed ABCG2. In drug-sensitive cells, inhibitors against hsa-miR-519c and hsa-miR-520h could augment the cytotoxic effect of mitoxantrone, suggesting a substantial role for both miRNAs in controlling ABCG2 level and thereby anticancer drug response. However, in drug-resistant cells, altering the levels of the two miRNAs did not have any effect on sensitivity to mitoxantrone. Taken together, these studies suggest that in ABCG2-overexpressing drug-resistant cells, hsa-miR-519c is unable to affect ABCG2 expression because the mRNA lacks its binding site, whereas hsa-miR-520h is sequestered and unable to limit ABCG2 expression. Given the recent observation that a truncated 3′UTR is also observed in ABCG2-overexpressing human embryonic stem cells, our results in drug-resistant cell lines suggest that 3′UTR truncation is a relatively common mechanism of ABCG2 regulation. [Mol Cancer Ther 2009;8(10):2959–68]

Supplementary Figures S1-S2; Supplementary Tables S1-S2.

PDF file - 47K, Effect of inhibition of autophagy on ABCG2 expression

Supplementary Figures 1-2 from Lapatinib (Tykerb, GW572016) Reverses Multidrug Resistance in Cancer Cells by Inhibiting the Activity of ATP-Binding Cassette Subfamily B Member 1 and G Member 2

PDF file - 26K, Chaperone expression after HDI treatment

Supplementary Table 1 from Sildenafil Reverses ABCB1- and ABCG2-Mediated Chemotherapeutic Drug Resistance

PDF file - 43K, HDIs do not induce endogenous ABCG2 expression in Flip-In-293 cells

PDF file - 52K, Ubiquitination state of ABCG2

PDF file - 65K

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PDF file - 29K, Impact of CHX and actinomycin D on HDI-induced ABCG2 surface overexpression

PDF file - 26K, CHX does not affect histone and �-tubulin acetylation induced by HDIs

Lapatinib is active at the ATP-binding site of tyrosine kinases that are associated with the human epidermal growth factor receptor (Her-1 or ErbB1) and Her-2. It is conceivable that lapatinib may inhibit the function of ATP-binding cassette (ABC) transporters by binding to their ATP-binding sites. The aim of this study was to investigate the ability of lapatinib to reverse tumor multidrug resistance (MDR) due to overexpression of ABC subfamily B member 1 (ABCB1) and ABC subfamily G member 2 (ABCG2) transporters. Our results showed that lapatinib significantly enhanced the sensitivity to ABCB1 or ABCG2 substrates in cells expressing these transporters, although a small synergetic effect was observed in combining lapatinib and conventional chemotherapeutic agents in parental sensitive MCF-7 or S1 cells. Lapatinib alone, however, did not significantly alter the sensitivity of non-ABCB1 or non-ABCG2 substrates in sensitive and resistant cells. Additionally, lapatinib significantly increased the accumulation of doxorubicin or mitoxantrone in ABCB1- or ABCG2-overexpressing cells and inhibited the transport of methotrexate and E217βG by ABCG2. Furthermore, lapatinib stimulated the ATPase activity of both ABCB1 and ABCG2 and inhibited the photolabeling of ABCB1 or ABCG2 with [125I]iodoarylazidoprazosin in a concentration-dependent manner. However, lapatinib did not affect the expression of these transporters at mRNA or protein levels. Importantly, lapatinib also strongly enhanced the effect of paclitaxel on the inhibition of growth of the ABCB1-overexpressing KBv200 cell xenografts in nude mice. Overall, we conclude that lapatinib reverses ABCB1- and ABCG2-mediated MDR by directly inhibiting their transport function. These findings may be useful for cancer combinational therapy with lapatinib in the clinic. [Cancer Res 2008;68(19):7905–14]

Supplementary Figure 1 from Sildenafil Reverses ABCB1- and ABCG2-Mediated Chemotherapeutic Drug Resistance

Ferroptosis is a form of cell death caused by direct or indirect inhibition of glutathione peroxidase 4 that leads to lethal lipid peroxidation. Several small molecule ferroptosis inducers (FINs) have been reported, yet little information is available regarding resistance mechanisms, particularly their interaction with the ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp, ABCB1) and ABCG2. Given the role that ABC transporters play in absorption, distribution, and excretion of many drugs, characterizing these interactions could provide information regarding oral bioavailability and brain penetration and may predict drug-drug interactions. Using ferroptosis-sensitive A673 cells transfected to express P-gp or ABCG2, we found that P-gp overexpression was able to confer resistance to FIN56 and the erastin derivatives imidazole ketone erastin and piperazine erastin. Results were confirmed with OVCAR8-derived NCI/ADR-RES cells that overexpress P-gp, where the P-gp inhibitor valspodar completely inhibited resistance to the FINs. P-gp-mediated resistance to imidazole ketone erastin and piperazine erastin was also reversed in UO-31 renal cancer cells by CRISPR-mediated knockout ofABCB1. At a concentration of 10 μM, the FINs ML-162, GPX inhibitor 26a, and PACMA31 were able to increase intracellular rhodamine 123 fluorescence over 10-fold in P-gp-expressing MDR-19 cells and GPX inhibitor 26a was able to increase intracellular purpurin-18 fluorescence over 4-fold in ABCG2-expressing R-5 cells. Expression of P-gp may reduce the efficacy of these FINs in cancers that express the transporter and may prevent access to sanctuary sites such as the brain. The ability of some FINs to inhibit P-gp and ABCG2 suggests potential drug-drug interactions.SIGNIFICANCE STATEMENTWhile several small-molecule ferroptosis inducers have been described, little work has addressed potential interactions with ABC transporters such as P-glycoprotein or ABCG2 that might limit bioavailability or brain penetration. We find that the ferroptosis inducers FIN56, imidazole ketone erastin, and piperazine erastin are substrates of P-glycoprotein. ML-162, GPX inhibitor 26a, and PACMA31 were found to inhibit P-glycoprotein, while GPX inhibitor 26a was additionally able to inhibit ABCG2, suggesting the potential for drug-drug interactions.

There is a need for simple, fast, and memory-efficient multidimensional data structures for dense and sparse storage that arise with numerical methods and in software applications. The data structures must perform equally well across multiple computer architectures, including CPUs and GPUs. For this purpose, we developed MATAR, a C++ software library that allows for simple creation and use of intricate data structures that is also portable across disparate architectures using Kokkos. The performance aspect is achieved by forcing contiguous memory layout (or as close to contiguous as possible) for multidimensional and multi-size dense or sparse MATrix and ARray (hence, MATAR) types. Our results show that MATAR has the capability to improve memory utilization, performance, and programmer productivity in scientific computing. This is achieved by fitting more work into the available memory, minimizing memory loads required, and by loading memory in the most efficient order. This document describes the purpose of the work, the implementation of each of the data types, and the resulting performance both in some simple baseline test cases and in an application code.

Histone deacetylase inhibitors (HDACis) are part of a growing class of epigenetic therapies used for the treatment of cancer. While elevated levels of the efflux pump P-gp are associated within vitroresistance to romidepsin, this mechanism does not translate to the clinic. We developed a romidepsin-resistant cell line with a resistance mechanism independent of P-gp function that acts upstream of the deacetylation process. We found that expression of the methyltransferase METTL7A is necessary for resistance, and that expression of METTL7A in naïve cells can drive resistance to thiol-containing HDACis. We demonstrate that METTL7A can methylate romidesinin vitroand that the ability of METTL7A to drive resistance to thiol-containing HDACis can be blocked by the methyltransferase inhibitor DCMB. Our data supports a model whereby exposure of cells to romidepsin selects for upregulation of the methyltransferase METTL7A, which in turn modifies the zinc-binding thiol, inactivating the drug.

The advancements in modern computer architecture have made it possible for the simulation of large-scale problems that are of industrial application. However, it can be time-intensive to continually port scientific codes to the ever-evolving computer architectures. Therefore, it is important to develop computational tools that make it easy for researchers to port their existing scientific software or develop new software that can run optimally on current and future computer architectures from different vendors. Also, it often advantageous for scientific software to leverage fine-grained parallelism on homogeneous architectures that are comprised of multi-core CPUs and on heterogeneous computer architectures comprised of multi-core CPUs and GPUs. This paper demonstrates the incorporation of performance portability and data-oriented design paradigms in the phase-field modeling of microstructure evolution. To achieve this, we utilized MATAR, a C+ + software library that allows the straightforward creation and usage of multidimensional and multi-size dense or sparse matrix and array data structures that are also portable across disparate architectures using Kokkos. As a case study, we use the phase-field model for spinodal decomposition which numerically solves the Cahn-Hilliard equation using the semi-implicit Fourier spectral method. Performance portability across multi-core CPUs and different GPU architectures are investigated. Additionally, numerical accuracy and performance gains between single and double precision phase-field calculations are investigated.

Enhanced-precision global sums are key to reproducibility in exascale applications. We examine two classic summation algorithms and show that vectorized versions are fast, good and reproducible at exascale. Both 256-bit and 512-bit implementations speed up the operation by almost a factor of four over the serial version. They thus demonstrate improved performance on global summations while retaining the numerical reproducibility of these methods.

RAS-mutant cancers are among the most refractory to treatment. Apart from new G12C genotype targeted therapies, strategies to kill RAS-mutant cells by directly targeting RAS or its downstream effectors have been mostly unsuccessful, mainly due to pathway redundancy and heterogeneities in RAS-induced phenotypes. Here we identified a RAS-phenotype that can be targeted by the histone deacetylase inhibitor (HDACi) romidepsin. We showed that the hyperacetylation induced by romidepsin depleted acetyl-CoA, the cell donor substrate for acetylation, and led to metabolic stress and death in KRAS-activated cells. Elastic net analysis on transcriptomics from a 608-cell panel confirmed that HDACi sensitivity was linked to a difference in profiles in two pathways involved in acetyl-CoA metabolism. The analysis of a clinical dataset confirmed that perturbation of the two acetyl-CoA pathways were correlated with HDACi sensitivity in patients treated with belinostat. Our analysis suggests the potential utility of a RAS-associated acetyl-CoA phenotype to sharpen treatment choices for RAS-activated tumors.

Given its similarities with mammalian systems, the zebrafish has emerged as a potential model to study the blood-brain barrier (BBB). Capillary endothelial cells at the human BBB express high levels of P-glycoprotein (P-gp, encoded by the ABCB1 gene) and ABCG2 (encoded by the ABCG2 gene). However, little information has been available about ATP-binding cassette transporters expressed at the zebrafish BBB. In this study, we focus on the characterization and tissue localization of two genes that are similar to human ABCB1, zebrafish abcb4 and abcb5. Cytotoxicity assays with stably-transfected cell lines revealed that zebrafish Abcb5 cannot efficiently transport the substrates doxorubicin and mitoxantrone compared to human P-gp and zebrafish Abcb4. Additionally, zebrafish Abcb5 did not transport the fluorescent probes BODIPY-ethylenediamine or LDS 751, while they were readily transported by Abcb4 and P-gp. A high-throughput screen conducted with 90 human P-gp substrates confirmed that zebrafish Abcb4 has overlapping substrate specificity with P-gp. Basal ATPase activity of zebrafish Abcb4 and Abcb5 was comparable to that of human P-gp. In the brain vasculature, RNAscope probes to detect abcb4 colocalized with staining by the P-gp antibody C219, while abcb5 was not detected. Zebrafish abcb4 also colocalized with claudin-5 expression in brain endothelial cells. Abcb4 and Abcb5 had different tissue localizations in multiple zebrafish tissues, consistent with different functions. The data suggest that zebrafish Abcb4 most closely phenocopies P-gp and that the zebrafish may be a viable model to study the role of the multidrug transporter P-gp at the BBB.

Applications often require a fast, single-threaded search algorithm over sorted data, typical in table-lookup operations. We explore various search algorithms for a large number of search candidates over a relatively small array of logarithmically-distributed sorted data. These include an innovative hash-based search that takes advantage of floating point representation to bin data by the exponent. Algorithms that can be optimized to take advantage of SIMD vector instructions are of particular interest. We then conduct a case study applying our results and analyzing algorithmic performance with the EOSPAC package. EOSPAC is a table look-up library for manipulation and interpolation of SESAME equation-of-state data. Our investigation results in a couple of algorithms with better performance with a best case eight times speedup over the original EOSPAC Hunt-and-Locate implementation. Our techniques should generalize to other instances of search algorithms seeking to get a performance boost from vectorization.