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Summary Knowledge of the full target space of bioactive substances, approved and investigational drugs as well as chemical probes, provides important insights into therapeutic potential and possible adverse effects. The existing compound-target bioactivity data resources are often incomparable due to non-standardized and heterogeneous assay types and variability in endpoint measurements. To extract higher value from the existing and future compound target-profiling data, we implemented an open-data web platform, named Drug Target Commons (DTC), which features tools for crowd-sourced compound-target bioactivity data annotation, standardization, curation, and intra-resource integration. We demonstrate the unique value of DTC with several examples related to both drug discovery and drug repurposing applications and invite researchers to join this community effort to increase the reuse and extension of compound bioactivity data., Graphical Abstract, Highlights • DTC is a crowd-sourcing-based web platform to annotate drug-target bioactivity data • The open environment improves data harmonization for drug repurposing applications • DTC offers a comprehensive, reproducible, and sustainable bioactivity knowledge base, Tang et al. launches a novel crowd-sourcing effort to standardize the collection, management, curation, and annotation of the notoriously heterogeneous compound-target bioactivity measurements. The web-based community platform aims to provide the most comprehensive, reproducible, and sustainable bioactivity knowledge base for the end users.

The BCR-ABL1 fusion gene is a driver oncogene in chronic myeloid leukaemia and 30-50% of cases of adult acute lymphoblastic leukaemia. Introduction of ABL1 kinase inhibitors (for example, imatinib) has markedly improved patient survival, but acquired drug resistance remains a challenge. Point mutations in the ABL1 kinase domain weaken inhibitor binding and represent the most common clinical resistance mechanism. The BCR-ABL1 kinase domain gatekeeper mutation Thr315Ile (T315I) confers resistance to all approved ABL1 inhibitors except ponatinib, which has toxicity limitations. Here we combine comprehensive drug sensitivity and resistance profiling of patient cells ex vivo with structural analysis to establish the VEGFR tyrosine kinase inhibitor axitinib as a selective and effective inhibitor for T315I-mutant BCR-ABL1-driven leukaemia. Axitinib potently inhibited BCR-ABL1(T315I), at both biochemical and cellular levels, by binding to the active form of ABL1(T315I) in a mutation-selective binding mode. These findings suggest that the T315I mutation shifts the conformational equilibrium of the kinase in favour of an active (DFG-in) A-loop conformation, which has more optimal binding interactions with axitinib. Treatment of a T315I chronic myeloid leukaemia patient with axitinib resulted in a rapid reduction of T315I-positive cells from bone marrow. Taken together, our findings demonstrate an unexpected opportunity to repurpose axitinib, an anti-angiogenic drug approved for renal cancer, as an inhibitor for ABL1 gatekeeper mutant drug-resistant leukaemia patients. This study shows that wild-type proteins do not always sample the conformations available to disease-relevant mutant proteins and that comprehensive drug testing of patient-derived cells can identify unpredictable, clinically significant drug-repositioning opportunities.

The Rho family of Ras superfamily small GTPases regulates a broad range of biological processes such as migration, differentiation, cell growth and cell survival. Therefore, the availability of small molecule modulators as tool compounds could greatly enhance research on these proteins and their biological function. To this end, we designed a biochemical, high throughput screening assay with complementary follow-up assays to identify small molecule compounds inhibiting MgcRacGAP, a Rho family GTPase activating protein involved in cytokinesis and transcriptionally upregulated in many cancers. We first performed an in-house screen of 20,480 compounds, and later we tested the assay against 342,046 compounds from the NIH Molecular Libraries Small Molecule Repository. Primary screening hit rates were about 1% with the majority of those affecting the primary readout, an enzyme-coupled GDP detection assay. After orthogonal and counter screens, we identified two hits with high selectivity towards MgcRacGAP, compared with other RhoGAPs, and potencies in the low micromolar range. The most promising hit, termed MINC1, was then examined with cell-based testing where it was observed to induce an increased rate of cytokinetic failure and multinucleation in addition to other cell division defects, suggesting that it may act as an MgcRacGAP inhibitor also in cells.

Introduction Acute myeloid leukaemia (AML) is a malignancy characterised by impaired cell differentiation and uncontrollable accumulation of immature myeloid progenitor cells in the bone marrow. Although most patients respond to the first line chemotherapeutic treatment, the majority of patients eventually relapse and therefore the overall survival from AML remains very poor. A therapy based on inducing the immature leukemic cells to differentiate may turn AML into a curable disease, as has been shown in the case of Acute Promyelocytic Leukaemia (APL), the M3 subtype of AML. However, AML is a heterogeneous disease and we still lack knowledge of how different genetic alterations disturb normal hematopoiesis and how the cell differentiation blockade may be lifted in the various subtypes of AML. Thus, our aims are 1) to identify therapies reversing aberrant cell differentiation in AML by using flow cytometry-based high-throughput drug screening 2) to establish links between molecular subtypes of AML and responsiveness to identified cell differentiating therapies. Material and methods Our approach is to explore drug-induced cell differentiation in primary samples from AML patients and genetically engineered AML mouse models using high-throughput flow cytometry. Specifically, we characterise samples by their expressed cell differentiation-related cell surface markers with and without drug treatment to identify compounds capable of inducing cell differentiation ex vivo . The primary AML patient samples are also profiled by exome- and RNA-sequencing. Results and discussions Preliminary results with a Flt3/Npm1 –mutated mouse model suggest that several compounds elicit cell differentiation-state specific responses, some of which could be indicative of a dynamic shift towards cell differentiation. For example, treatment with mTOR-kinase inhibitors sapanisertib and vistusertib resulted in a concentration dependent increase of differentiating cells marked by CD11b expression and decrease of stem cells marked by CD34 expression. Conclusion We have established a high-throughput flow cytometric screening platform to identify drugs that are able to induce differentiation in primary AML samples and genetically engineered mouse models of AML. By integrating drug response data with genetic background information from samples we aim to reveal novel genotype-phenotype relationships in the level of cell differentiation that could eventually be translated into clinically relevant biomarkers and treatment options for AML patients.

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by impaired differentiation and uncontrollable proliferation of myeloid progenitor cells. Due to high relapse rates, overall survival for this rapidly progressing disease is poor. The significant challenge in AML treatment is disease heterogeneity stemming from variability in maturation state of leukemic cells of origin, genetic aberrations among patients, and existence of multiple disease clones within a single patient. Disease heterogeneity and the lack of biomarkers for drug sensitivity lie at the root of treatment failure as well as selective efficacy of AML chemotherapies and the emergence of drug resistance. Furthermore, standard-of-care treatment is aggressive, presenting significant tolerability concerns to the commonly advanced-age AML patient. In this review, we examine the concept and potential of molecular stratification, particularly with biologically relevant drug responses, in identifying low-toxicity precision therapeutic combinations and clinically relevant biomarkers for AML patient care as a way to overcome these challenges in AML treatment.

Ras-mediated transformation is associated with upregulation of cyclooxygenase-2 (COX-2), which in turn promotes prostaglandin E2 (PGE2) synthesis and secretion. Although recent studies have identified molecular mechanisms by which Ras mediates upregulation of COX-2, conflicting observations have been made. Furthermore, while COX-2 upregulation has been shown to be important for Ras transformation, the signaling pathways initiated by PGE2-stimulation of EP family of heterotrimeric G protein-coupled receptors (GPCR) and contribution of PGE2 signaling to Ras-mediated transformation are issues that remain unresolved. In this study, we first determined that Raf effector pathway activation of the extracellular-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) cascade alone was sufficient and necessary for COX-2 and PGE2 upregulation. However, Raf-independent regulation of the c-jun N-terminal kinase (JNK) and p38 MAPK cascades is also involved in COX-2 and PGE2 upregulation, with the JNK and p38 pathways exhibiting opposing roles in COX-2 and PGE2 upregulation. Furthermore, in contrast to previous studies, we found that an epidermal growth factor (EGF) receptor autocrine growth mechanism, another Raf-independent signaling mechanism, was necessary for COX-2 and PGE2 upregulation. Second, we determined that inhibition of EP1/2 receptor function blocked growth transformation by Ras, demonstrating that PGE2 upregulation is a key transforming function of COX-2. Finally, we found that PGE2 stimulated the activation of Ras and ERK, but not Akt, and reduced matrix deprivation-induced apoptosis, in untransformed epithelial cells. In summary, our studies define additional, multiple signaling mechanisms that promote COX-2 and PGE2 expression and show that COX-2-stimulated PGE2-EP receptor signaling is required for growth and survival transformation by Ras.

Although mutated forms of ras are not associated with the majority of breast cancers (

Serum immunoglobulin A (IgA) concentrations were determined in 12 600 adult Swedish twins, applying a high-throughput reverse-phase protein microarray technique. The prevalence of IgA deficiency (IgAD) was found to be 1:241 in monozygotic (MZ) twins and 1:198 in dizygotic (DZ) twins. Hence, the prevalence in twins is markedly elevated as compared with the normal Swedish adult population (1:600). The twins did not show a difference in the frequency of HLA haplotypes in comparison with almost 40 000 healthy Swedish controls. As expected, the risk-conveying HLA alleles A*01, B*08 and DRB1*01 were overrepresented among the IgAD twins and were also associated with significantly lower mean serum IgA concentrations in the twin cohort. In contrast, significantly higher mean IgA concentrations were found among individuals carrying the protective HLA alleles B*07 and DRB1*15. Exome sequencing data from two MZ twin pairs discordant for the deficiency showed no differences between the siblings. Model fitting analyses derived a heritability of 35% and indicate that genetic influences are modestly important for IgAD. The probandwise concordance rates for IgAD were found to be 31% for MZ and 13% for DZ twins.

Publisher Summary This chapter summarizes the general features of R-Ras proteins that are shared with Ras proteins, and then highlights unique features of each R-Ras protein. Ras proteins (H-Ras, K-Ras4A and 4B, and N-Ras) are regulators of signal transduction, mutated in 30 percent of human cancers, and targets for novel approaches for cancer treatment. Ras proteins are the founding members of a superfamily of small GTP binding and hydrolyzing proteins. The three R-Ras subfamily members, R-Ras, TC21/R-Ras2, and M-Ras/R-Ras3, mediate cell growth, division, differentiation, and death by utilizing both novel pathways and those regulated by Ras and other Ras-related proteins. Further, R-Ras subfamily members, especially R-Ras itself, contribute to cellular processes such as integrin-mediated cell adhesion in a manner distinct from that of Ras. Deciphering the full contribution of R-Ras, TC21, and M-Ras to cellular growth control clearly awaits further study.

The Ras-related, estrogen-regulated growth inhibitor (Rerg) is a Ras-related small GTPase and candidate tumor suppressor. Rerg gene expression is stimulated by the estrogen receptor alpha (ERalpha), and Rerg gene expression is absent in ER-negative breast cancers. ER-negative breast cancers are highly invasive and metastastic and are typically more advanced than their ER-positive counterparts. Like Ras, Rerg binds and hydrolyzes GTP, but unlike Ras, Rerg has been shown to possess growth inhibitory activity in breast cancer cells. The precise role that Rerg loss plays in breast cancer growth and the mechanisms by which it does so are unknown. This chapter describes tools used to detect and manipulate the expression of Rerg in breast cancer cells. We validate use of an antibody to detect Rerg expression. We describe the generation of expression vectors that encode wild-type and mutants of Rerg that are altered in GDP/GTP regulation. We also describe the development of an inducible Rerg expression system and of a retrovirus-based RNA interference approach to repress Rerg expression. These tools will be invaluable in evaluating the biological function of Rerg in breast cancer.

The Ras-related, estrogen-regulated growth inhibitor (Rerg) is a Ras-related small GTPase and candidate tumor suppressor. Rerg gene expression is stimulated by the estrogen receptor alpha (ERalpha), and Rerg gene expression is absent in ER-negative breast cancers. ER-negative breast cancers are highly invasive and metastastic and are typically more advanced than their ER-positive counterparts. Like Ras, Rerg binds and hydrolyzes GTP, but unlike Ras, Rerg has been shown to possess growth inhibitory activity in breast cancer cells. The precise role that Rerg loss plays in breast cancer growth and the mechanisms by which it does so are unknown. This chapter describes tools used to detect and manipulate the expression of Rerg in breast cancer cells. We validate use of an antibody to detect Rerg expression. We describe the generation of expression vectors that encode wild-type and mutants of Rerg that are altered in GDP/GTP regulation. We also describe the development of an inducible Rerg expression system and of a retrovirus-based RNA interference approach to repress Rerg expression. These tools will be invaluable in evaluating the biological function of Rerg in breast cancer.

The T315I gatekeeper mutation confers resistance to majority of approved ABL1 inhibitors, with only ponatinib demonstrating efficacy in BCR-ABL1(T315I)-driven disease. However, vascular adverse events and selection of resistant compound mutations limit its clinical utility. Hence, there is an unmet need for novel therapies for patients with gatekeeper-mutated Ph+ leukemia. In this study we integrated comprehensive drug sensitivity testing with structural analysis to characterize the tyrosine kinase inhibitor axitinib as a putative novel therapy for BCR-ABL1(T315I)-driven leukemias. To address this we profiled BCR-ABL1(T315I)-driven CML/Ph+ ALL patient samples against 300 anti-cancer compounds (approved and investigational drugs). Ex vivo drug sensitivity testing of primary cells derived from a Ph+ ALL patient revealed a marked and cancer-selective response to the VEGFR inhibitor axitinib. Strikingly, axitinib exhibited higher sensitivity in T315I positive Ph+ patient samples in comparison to T315I negative CML and ALL patient samples. In line with the ex vivo drug response data, axitinib inhibited the kinase activity of ABL1(T315I) with similar potency as its primary target VEGFR2, while the potency to non-mutated ABL1 was 30-fold lower. Analogously, in engineered Ba/F3 cells, axitinib showed a 10-fold higher inhibition of T315I than non-mutated ABL1-driven autophosphorylation and cell growth. To better understand the molecular mechanisms of the BCR-ABL1(T315I) selectivity, we solved the crystal structure of axitinib in complex with ABL1(T315). The structure revealed that axitinib bound to a mutation induced active conformation of ABL1(T315I), different than the binding mode in non-mutated ABL1, likely explaining the increased potency towards ABL1(T315I). Moreover, axitinib occupied a distinct binding space than all approved ABL1 inhibitors, signifying that axitinib will have a unique mutation vulnerability profile. Compassionate two week treatment of a CML patient harboring the T315I mutation with axitinib, resulted in a 5-fold reduction of T315I transcript levels in the bone marrow, further suggesting that axitinib can produce specific and effective responses in patients with BCR-ABL1(T315I)-driven leukemia. In conclusion, we demonstrate that axitinib potently inhibits BCR-ABL1(T315I) via a gatekeeper mutant-selective mechanism. Since axitinib is in clinical use for treatment of refractory renal cell carcinoma with a manageable safety profile, our data provide a sound basis for readily repurposing axitinib for BCR-ABL1(T315I)-driven leukemia. Finally, the distinct mechanism of inhibition by axitinib serves as an exemplar for development of even more effective gatekeeper-mutant selective inhibitors targeting ABL1 as well as other clinically important kinases, such as EGFR and KIT. Citation Format: Tea Pemovska, Eric Johnson, Mika Kontro, Gretchen A. Repasky, Jeffrey Chen, Peter Wells, Ciarán N. Cronin, Michele McTigue, Olli Kallioniemi, Kimmo Porkka, Brion W. Murray, Krister Wennerberg. Axitinib targets gatekeeper-mutant BCR-ABL1(T315I)-driven leukemia in a distinct and selective fashion. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 676. doi:10.1158/1538-7445.AM2015-676

RERG (Ras-related and estrogen-regulated growth inhibitor), a gene that encodes a small GTP binding and hydrolyzing protein (GTPase) of the Ras superfamily, was originally identified in gene microarray analysis as a gene of which expression is down-regulated in estrogen receptor (ER)-negative breast tumors. Subsequently, RERG mRNA was detected in ER-positive breast tumor-derived cell lines, but not in any of the ER-negative cell lines examined. Furthermore, a comparison of matched tumor and normal tissue samples suggests that RERG expression is lost in kidney, breast, ovary, and colon tumors. The lack of RERG expression in many highly aggressive breast carcinomas suggests that RERG plays an inhibitory role in cell growth and division. In fact, growth of breast tumor cells was inhibited by overexpression of RERG both in vitro and in vivo. In this chapter, we summarize the reagents and approaches used to characterize RERG gene expression, to demonstrate that RERG functions as a GTP/GDP molecular switch, and to characterize the growth inhibitory activity of RERG.

Ras proteins activate cytoplasmic signaling cascades that mediate responses in growth, cellular differentiation, and survival. Therefore, it is not surprising that mutationally activated Ras proteins have been found in many human cancers. Determining the effector protein signaling pathways through which Ras causes cellular transformation is important for creating targeted therapeutics that will specifically block the oncogenic effects of activated Ras. In 1993, Raf serine/threonine kinases were identified as key downstream effectors of Ras signaling and transformation. While Raf remains the best characterized Ras effector, the rapid expansion of the Ras effector pool has demonstrated that Ras transforming activity is also mediated by Raf-independent effector signaling pathways. These include phosphatidylinositol 3-kinase and phospholipase regulators of phospholipid metabolism, and guanine nucleotide exchange factors and activators of Ras-related proteins. Further complexity arose when a new and seemingly incongruous group of pro-apoptotic Ras effectors with tumor suppressor function was identified. This chapter will summarize recent findings of mutational activation of B-Raf in human cancers and examine the importance of non-Raf effectors in Ras-mediated signaling and transformation

RERG (Ras-related and estrogen-regulated growth inhibitor), a gene that encodes a small GTP binding and hydrolyzing protein (GTPase) of the Ras superfamily, was originally identified in gene microarray analysis as a gene of which expression is down-regulated in estrogen receptor (ER)-negative breast tumors. Subsequently, RERG mRNA was detected in ER-positive breast tumor-derived cell lines, but not in any of the ER-negative cell lines examined. Furthermore, a comparison of matched tumor and normal tissue samples suggests that RERG expression is lost in kidney, breast, ovary, and colon tumors. The lack of RERG expression in many highly aggressive breast carcinomas suggests that RERG plays an inhibitory role in cell growth and division. In fact, growth of breast tumor cells was inhibited by overexpression of RERG both in vitro and in vivo. In this chapter, we summarize the reagents and approaches used to characterize RERG gene expression, to demonstrate that RERG functions as a GTP/GDP molecular switch, and to characterize the growth inhibitory activity of RERG.

Ras proteins function as signal transducers and are mutationally activated in many human cancers. In 1993, Raf was identified as a key downstream effector of Ras signaling, and it was believed then that the primary function of Ras was simply to facilitate Raf activation. However, the subsequent discovery of other proteins that are effectors of Ras function suggested that oncogenic activities of Ras are mediated by both Raf-dependent and Raf-independent signaling. Further complexity arose with the identification of Ras effectors with putative tumor suppressor, rather than oncogenic, functions. However, the recent identification of B-raf mutations in human cancers has renewed the debate regarding whether Raf activation alone promotes Ras-mediated oncogenesis. In this article, we summarize the current knowledge of the contribution of Ras effectors in Ras-mediated oncogenesis.

The use of ABL1 kinase inhibitors has dramatically improved the outcome for chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients. However, resistance after treatment still poses a major clinical challenge. The most common resistance mechanism following treatment with first or second line therapy is the occurrence of a T315I mutation in the kinase domain of BCR-ABL1. Only one clinically available ABL1 inhibitor, ponatinib, has been shown to target this gatekeeper mutation, but has recently been associated with significant adverse effects. Hence, there is an unmet need for new and improved therapies for patients with T315I BCR-ABL1 leukemias. In this study we set out to functionally and molecularly profile BCR-ABL1 T315I-driven CML/Ph+ALL patient samples to understand the disease pathogenesis and identify novel therapies with a drug sensitivity and resistance testing (DSRT) platform covering 306 approved and investigational oncology compounds. Mononuclear cells isolated from patient bone marrow were plated with drugs on 384-well plates. Each compound was tested for its effect on cell growth and survival in a 10,000-fold concentration range enabling the generation of dose response curves and by comparing to healthy donor mononuclear cells, selective drug sensitivity scores (sDSS). Ex vivo DSRT results of one CML and two Ph+ALL patient samples with the T315I mutation revealed a marked and specific sensitivity (IC50 30-40nM) to the tyrosine kinase inhibitor axitinib, originally developed as a VEGFR inhibitor. Strikingly, sensitivity to axitinib was higher in these T315I positive patient samples than in T315I negative CML or ALL patient samples or any other leukemic samples. Supporting the notion that axitinib is a direct T315I BCR-ABL1 inhibitor we observed that Ba/F3 cells transformed with T315I BCR-ABL1 were sensitive to axitinib while the same cells transformed with wild type BCR-ABL1 were not. Finally, we discovered that axitinib has been described to have selective binding towards T315I ABL1 compared to the wild type kinase (Kd 1.5 nM vs. 36 nM, respectively, Davis et al. 2011, Nat. Biotechnol. 29:1046-1051). Based on this information, the T315I CML patient mentioned above was compassionately treated with axitinib for 2 weeks resulting in a rapid 4-fold reduction of the mutated transcript levels in blood suggesting targeted in vivo activity of the drug. In summary, we demonstrated that axitinib is a potent BCR-ABL1 T315I inhibitor both in vitro and in vivo. In light to the fact that axitinib is currently approved as a second line therapy for renal cell carcinoma and is well tolerated in patients, there is an opportunity to repurpose axitinib for Ph+ leukemia patients with T315I mutations with significantly shorter clinical development time. Citation Format: Tea Pemovska, Mika Kontro, Gretchen A. Repasky, Kimmo Porkka, Krister Wennerberg. The tyrosine kinase inhibitor axitinib targets T315I gatekeeper-mutant Philadelphia chromosome-positive leukemias in vitro and in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4763. doi:10.1158/1538-7445.AM2014-4763

Mutated and constitutively activated Ras oncoproteins are found in ∼33% of all human cancers. In particular, Ras mutations are found in a majority of pancreatic cancers (90%), and aberrant Ras activation has been shown to be critical for promoting pancreatic adenocarcinomas. Ras-mediated transformation is associated with downregulation of Pigment Epithelium Derived Factor (PEDF), an anti-angiogenic and lipid regulatory factor shown to suppress oncogenic K-Ras-induced invasive disease in vivo. PEDF has also been shown to have anti-tumorigenic activity on tumor cells themselves. Specifically, mice with chronically active mutant KRAS(12D) background form precancerous lesions with high frequency, and knockout of PEDF expression specifically in pancreatic tissue resulted in a significant aggravation of the precancerous phenotype. The mechanism(s) of this biological phenotype is unclear and the specific role of PEDF as a potential tumor suppressor gene in Ras-mediated oncogenesis remains unresolved. Western analyses were used in this study to investigate the expression of PEDF in K-Ras-transformed and nontransformed human pancreatic duct-derived cells (HPNE) and their surrounding cell culture growth media. We observe that PEDF is upregulated both intracellularly under growth and serum-starved conditions and extracellularly under serum-starved conditions in K-Ras-transformed human pancreatic cells. We are also investigating the role of exogenous PEDF in cell proliferation induced by oncogenic variants of K-Ras. While no significant difference was observed in cell proliferation between the PEDF-treated and nontreated cells (whether they be K-Ras-transformed and nontransformed cells) in growth conditions, a decrease in cell proliferation was observed in the PEDF-treated K-Ras-transformed cells under serum-starved conditions. By better understanding the link between Ras and PEDF we will be able to assess the contribution and therapeutic potential of PEDF to formation and progression of pancreatic cancer while investigating novel targets for therapeutic intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3129.