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Supplementary Table 3. Genetic variants associated with 5-year PFS

Supplementary Table 2. Genetic variants associated with histologic response

Purpose: Overall survival in patients with osteosarcoma is only 60%. Poor response to chemotherapy is the dominant risk factor for poor survival. Pharmacogenetic research can offer possibilities to optimize treatment and improve outcome. We applied a pathway-based approach to evaluate the cumulative effect of genes involved in the metabolism of cisplatin and doxorubicin in relationship to clinical outcome.Experimental Design: We included 126 patients with osteosarcoma. To comprehensively assess common genetic variation in the 54 genes selected, linkage disequilibrium (LD; r2 = 0.8)–based tag-single nucleotide polymorphisms (SNP) strategy was used. A final set of 384 SNPs was typed using Illumina Beadarray platform. SNPs significantly associated with 5-year progression-free survival (PFS) were replicated in another 64 patients with osteosarcoma.Results: We identified five variants in FasL, MSH2, ABCC5, CASP3, and CYP3A4 that were associated with 5-year PFS. Risk stratification based on the combined effects of the risk alleles showed a significant improvement of 5-year PFS. Patients that carried no or only one risk allele had a 5-year PFS of 100% compared with a 5-year PFS of 84.4% for carriers of two or three risk alleles, 66.7% PFS if a patient carried four to five alleles, and a 5-year PFS of 41.8% for patients with >5 risk alleles (P < 0.001).Conclusions: We identified several genes that showed association with PFS in patients with osteosarcoma. These pharmacogenetic risk factors might be useful to predict treatment outcome and to stratify patients immediately after diagnosis and offer the possibility to improve treatment and outcome. Clin Cancer Res; 21(15); 3436–41. ©2015 AACR.

Supplementary Table 1. Set of 381 SNPs

PDF file - 114K, Uncropped immunoblots and autoradiograms

PDF file - 643K, Rho-kinase inhibition rescues contractile and migratory phenotype of TRPM7 shRNA cells

PDF file - 54K, TRPM7 shRNA#2 treatment reduces malignant phenotype of MDA-MB-231 cells

PDF file - 119K, TRPM7 expression measurements on TRPM7 knockdown MDA-MB-231 and MCF7 cells and TRPM7 rescued cells

PDF file - 92K, Patient- and tumor characteristics in discovery and validation cohort

PDF file - 3.9MB, TRPM7 knockdown does not affect MDA-MB-231 cell proliferation but impairs metastasis formation in vivo

TRPM7 encodes a Ca2+-permeable nonselective cation channel with kinase activity. TRPM7 has been implicated in control of cell adhesion and migration, but whether TRPM7 activity contributes to cancer progression has not been established. Here we report that high levels of TRPM7 expression independently predict poor outcome in breast cancer patients and that it is functionally required for metastasis formation in a mouse xenograft model of human breast cancer. Mechanistic investigation revealed that TRPM7 regulated myosin II–based cellular tension, thereby modifying focal adhesion number, cell–cell adhesion and polarized cell movement. Our findings therefore suggest that TRPM7 is part of a mechanosensory complex adopted by cancer cells to drive metastasis formation. Cancer Res; 72(16); 4250–61. ©2012 AACR.

PDF file - 96K, TRPM7 expression levels and clinical parameters

Contains fulltext : 241347.pdf (Publisher’s version ) (Closed access) Asparaginase (ASNase) therapy has been a mainstay of acute lymphoblastic leukemia (ALL) protocols for decades and shows promise in the treatment of a variety of other cancers. To improve the efficacy of ASNase treatment, we used a CRISPR/Cas9-based screen to identify actionable signaling intermediates that improve the response to ASNase. Both genetic inactivation of Bruton's tyrosine kinase (BTK) and pharmacological inhibition by the BTK inhibitor ibrutinib strongly synergize with ASNase by inhibiting the amino acid response pathway, a mechanism involving c-Myc-mediated suppression of GCN2 activity. This synthetic lethal interaction was observed in 90% of patient-derived xenografts, regardless of the genomic subtype. Moreover, ibrutinib substantially improved ASNase treatment response in a murine PDX model. Hence, ibrutinib may be used to enhance the clinical efficacy of ASNase in ALL. This trial was registered at www.clinicaltrials.gov as # NCT02884453.

Minimal residual disease (MRD) diagnostics are implemented in most clinical protocols for patients with acute lymphoblastic leukaemia (ALL) and are mostly performed using rearranged immunoglobulin (IG) and/or T-cell receptor (TR) gene rearrangements as molecular polymerase chain reaction targets. Unfortunately, in 5–10% of patients no or no sensitive IG/TR targets are available, and patients therefore cannot be stratified appropriately. In the present study, we used fusion genes and genomic deletions as alternative MRD targets in these patients, which retrospectively revealed appropriate MDR stratification in 79% of patients with no (sensitive) IG/TR target, and a different risk group stratification in more than half of the cases.

Contains fulltext : 234011.pdf (Publisher’s version ) (Open Access) Targeting tumor cell metabolism is an attractive form of therapy, as it may enhance treatment response in therapy resistant cancers as well as mitigate treatment-related toxicities by reducing the need for genotoxic agents. To meet their increased demand for biomass accumulation and energy production and to maintain redox homeostasis, tumor cells undergo profound changes in their metabolism. In addition to the diversion of glucose metabolism, this is achieved by upregulation of amino acid metabolism. Interfering with amino acid availability can be selectively lethal to tumor cells and has proven to be a cancer specific Achilles' heel. Here we review the biology behind such cancer specific amino acid dependencies and discuss how these vulnerabilities can be exploited to improve cancer therapies.

Although long-term survival in pediatric acute lymphoblastic leukemia (ALL) currently exceeds 90%, some subgroups, defined by specific genomic aberrations, respond poorly to treatment. We previously reported that leukemias harboring deletions or mutations affecting the B-cell transcription factor

Contains fulltext : 248373.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 283489.pdf (Publisher’s version ) (Open Access) Although long-term survival in pediatric acute lymphoblastic leukemia (ALL) currently exceeds 90%, some subgroups, defined by specific genomic aberrations, respond poorly to treatment. We previously reported that leukemias harboring deletions or mutations affecting the B-cell transcription factor IKZF1 exhibit a tumor cell intrinsic resistance to glucocorticoids (GCs), one of the cornerstone drugs used in the treatment of ALL. Here, we identified increased activation of both AKT and ERK signaling pathways as drivers of GC resistance in IKZF1-deficient leukemic cells. Indeed, combined pharmacological inhibition of AKT and ERK signaling effectively reversed GC resistance in IKZF1-deficient leukemias. As inhibitors for both pathways are under clinical investigation, their combined use may enhance the efficacy of prednisolone-based therapy in this high-risk patient group.

Contains fulltext : 248362.pdf (Publisher’s version ) (Open Access) INTRODUCTION: One-quarter of the relapses in children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) occur very early (within 18 months, before completion of treatment), and prognosis in these patients is worse compared to cases that relapse after treatment has ended. METHODS: In this study, we performed a genomic analysis of diagnosis-relapse pairs of 12 children who relapsed very early, followed by a deep-sequencing validation of all identified mutations. In addition, we included one case with a good initial treatment response and on-treatment relapse at the end of upfront therapy. RESULTS: We observed a dynamic clonal evolution in all cases, with relapse almost exclusively originating from a subclone at diagnosis. We identified several driver mutations that may have influenced the outgrowth of a minor clone at diagnosis to become the major clone at relapse. For example, a minimal residual disease (MRD)-based standard-risk patient with ETV6-RUNX1-positive leukemia developed a relapse from a TP53-mutated subclone after loss of the wildtype allele. Furthermore, two patients with TCF3-PBX1-positive leukemia that developed a very early relapse carried E1099K WHSC1 mutations at diagnosis, a hotspot mutation that was recurrently encountered in other very early TCF3-PBX1-positive leukemia relapses as well. In addition to alterations in known relapse drivers, we found two cases with truncating mutations in the cohesin gene RAD21. CONCLUSION: Comprehensive genomic characterization of diagnosis-relapse pairs shows that very early relapses in BCP-ALL frequently arise from minor subclones at diagnosis. A detailed understanding of the therapeutic pressure driving these events may aid the development of improved therapies.

Tyrosine kinase 2 (TYK2) is a member of the Janus kinase/signal transducer and activator of transcription pathway, which is central in cytokine signaling. Previously, germline TYK2 mutations have been described in two patients developing de novo T-cell acute lymphoblastic leukemias (T-ALLs) or precursor B-ALLs. The mutations (P760L and G761V) are located within the regulatory pseudokinase domain and lead to constitutive activation of TYK2. We demonstrate the transformation capacity of TYK2P760L in hematopoietic cell systems including primary bone marrow cells. In vivo engraftment of TYK2P760L-expressing cell lines led to development of leukemia. A kinase inhibitor screen uncovered that oncogenic TYK2 acts synergistically with the PI3K/AKT/mTOR and CDK4/6 pathways. Accordingly, the TYK2-specific inhibitor deucravacitinib (BMS986165) reduces cell viability of TYK2P760Ltransformed cell models and ex vivo cultured TYK2P760L-mutated patient-derived xenograft cells most efficiently when combined with mTOR or CDK4/6 inhibitors. Our study thereby pioneers novel treatment options for patients suffering from TYK2-driven acute leukemia.

Background: Adult mammalian cardiomyocytes (CM) are predominantly post-mitotic and cannot proliferatively repair the heart following myocardial infarction (MI). Overexpression of the transcription factor Tbx20 in adult mouse CMs promotes proliferative cardiac repair post-MI, via mechanisms including direct repression of anti-proliferative genes p21 , Meis1 , and Btg2 . Btg2 (B-cell translocation gene 2), a tumor suppressor and transcriptional co-regulator, exhibits high structural and functional similarity with Btg1. However, both Btg1 and Btg2 (Btg1/2) are virtually uncharacterized in the heart. Here, we investigate the role of Btg1/2 in postnatal cardiac maturation. Methods and Results: By immunostaining in embryonic, neonatal, and adult C57BL/6 mouse hearts, the highest expression of Btg1/2 was observed in late fetal and early neonatal ventricles, concurrent with upregulation of other CM cell cycle inhibitors. In neonatal mouse CMs in vitro, siRNA-mediated loss of Btg2 leads to increased CM proliferation. In vivo , Btg1/2 constitutive single- and double- knockout (SKO and DKO respectively) mice exhibit normal heart weight-to-body weight ratios compared to age-matched wildtype (WT) controls, at postnatal day (P)7, P30, and 1 year after birth. Interestingly, at P7, DKO mice have significantly higher CM mitotic activity, as indicated by pHH3 staining, compared to WT. In addition, DKO mice also exhibit significantly smaller CM cross-sectional area at P7 compared to WT. However, by P15, CM mitotic activity and cell size are comparable between WT and Btg1/2 KO mice. Currently, siRNA-mediated knockdown of Btg1/2 in neonatal rat ventricular cardiomyocyte cultures and RNAseq studies are being performed, to assess the transcriptional regulatory roles of Btg1/2 in rodent CMs. Conclusions: Here, we highlight two novel regulators of postnatal CM maturation, Btg1 and Btg2, which are upregulated coincident with CM mitotic arrest in mice. Similar to p21 and Meis1, Btg1/2 depletion in mice induces a brief period of increased CM proliferative activity before onset of CM cell cycle arrest. Our results provide evidence for Btg1/2 working in tandem with other cardiac transcription factors and cell cycle regulators, to control CM mitotic arrest after birth.

Supplemental Digital Content is available in the text, Genomic alterations in relapsed B-cell precursor acute lymphoblastic leukemia (BCP-ALL) may provide insight into the role of specific genomic events in relapse development. Along this line, comparisons between the spectrum of alterations in relapses that arise in different upfront treatment protocols may provide valuable information on the association between the tumor genome, protocol components and outcome. Here, we performed a comprehensive characterization of relapsed BCP-ALL cases that developed in the context of 3 completed Dutch upfront studies, ALL8, ALL9, and ALL10. In total, 123 pediatric BCP-ALL relapses and 77 paired samples from primary diagnosis were analyzed for alterations in 22 recurrently affected genes. We found pronounced differences in relapse alterations between the 3 studies. Specifically, CREBBP mutations were observed predominantly in relapses after treatment with ALL8 and ALL10 which, in the latter group, were all detected in medium risk-treated patients. IKZF1 alterations were enriched 2.2-fold (p = 0.01) and 2.9-fold (p

Neuroblastoma is the most common extracranial solid tumor in children and originates from poorly differentiated neural crest progenitors. High-risk neuroblastoma patients frequently present with metastatic disease at diagnosis. Despite intensive treatment, patients often develop refractory disease characterized by poorly differentiated, therapy resistant cells. Although adjuvant therapy using retinoic acid (RA)-induced differentiation may increase event-free survival, in the majority of cases response to RA-therapy is inadequate. Consequently, current research aims to identify novel therapeutic targets that enhance the sensitivity to RA and induce neuroblastoma cell differentiation. The similarities between neural crest development and neuroblastoma progression provide an appealing starting point. During neural crest development the EMT-transcription factor SNAI2 plays an important role in neural crest specification as well as neural crest cell migration and survival. Here, we report that CRISPR/Cas9 mediated deletion as well as shRNA mediated knockdown of the EMT-transcription factor SNAI2 promotes cellular differentiation in a variety of neuroblastoma models. By comparing mRNA expression data from independent patient cohorts, we show that a SNAI2 activity-based gene expression signature significantly correlates with event-free survival. Loss of SNAI2 function reduces self-renewal, 3D invasion as well as metastatic spread in vivo, while strongly sensitizing neuroblastoma cells to RA-induced growth inhibition. Together, our data demonstrate that SNAI2 maintains progenitor-like features in neuroblastoma cells while interfering with RA-induced growth inhibition. We propose that targeting gene regulatory circuits, such as those controlling SNAI2 function, may allow reversion of RA-therapy resistant neuroblastoma cells to a more differentiated and therapy responsive phenotype.

Background: MLL (KMT2A)-rearranged acute lymphoblastic leukemia (MLLr ALL) is a rare but aggressive subset that represents 5% of childhood ALL cases, and accounts for about 70% of infant leukemias. While overall survival in these young children is around 50%, after relapse, MLLr ALL becomes an almost incurable disease, highlighting the urgent clinical need for new strategies for this patient group. The histone methyl transferase function of the MLL fusion protein complex requires the methionine metabolite s-adenosylmethionine (SAM) as methyl donor, suggesting a selective sensitivity of MLL-r ALL for perturbations in methionine availability. Recent studies in solid tumor models suggest clinical utility of methionine restricted diets or oral administration of methionine depleting enzyme Methionine Gamma Lyase (MGL) to be safe and effective. Therefore, we explored the effect of methionine restriction (MR) as a potential, new therapy for MLLr ALL. Methods: We compared the effects of MR on metabolic activity and viability between MLLr and non-MLLr pre-BCP ALL cell lines using enzymatic depletion, small molecule inhibitors targeting methionine metabolism, and restrictive culture conditions. To identify intrinsic metabolic differences between MLLr and non-MLLr cells and explore how MR impinges on their metabolic state, we performed global metabolomics on MLLr SEM cells and non-MLLr NALM6 cells cultured with complete depletion of methionine. Additionally, we used RNA sequencing to assess the global effects of MR on gene expression, and a CRISPR/Cas9-based reverse genetic screen to identify sensitizers towards MR. Results were validated in vitro using targeted knockouts and small-molecule inhibitors, as well as in vivo using a 95% methionine restricted diet. Immunocompromised mice were engrafted with MLLr SEM cells and 7 days after transplantation, mice were randomized to control or 95% MR diet. Leukemia progression was monitored by flowcytometric detection of human lymphocytes in the blood. Results: We observed that depletion of methionine reduces metabolic activity in almost all BCP-ALL (B-ALL) cell lines, however, only in MLLr B-ALL cell lines was rapid apoptosis induced (Figure 1A). Global metabolic profiling revealed significant basal metabolic differences, of note being SAM, whose levels were approximately 5-fold higher in MLLr SEM cells compared to non-MLLr NALM6 cells. Consistent with this, addition of SAM completely rescued MLLr cell lines from methionine depletion induced apoptosis, an effect not observed in non-MLLr cells (Figure 1A). Metabolomic profiling also highlighted different salvage mechanisms at play in NALM6 cells, with the folate cycle and polyamine synthesis pathway being activated upon MR. Together, these results indicate that MLLr B-ALL cells are selectively sensitive to MR. In line with this, RNASeq data showed significant decreased expression of several known MLL fusion target genes such as PROM1, HOXA10, and MEIS1 in response to MR. To obtain further insight into the pathways involved in the response to MR and to identify potential therapeutic targets that further sensitize cells to MR, we performed a CRISPR/Cas9-based screen. This identified three members of the Bromodomain- and extra-terminal domain (BET) family as potential modifiers of the response to MR in SEM cells. Indeed, RNAseq analysis showed that Myc activity as a proxy of BRD4 function, was strongly suppressed by MR. Finally, preliminary results show the efficacy of dietary intervention alone on leukemia progression. We observe with 95% MR diet, significant delays on leukemic growth (Figure 1B). Moreover, the MR diet was well tolerated, as indicated by minimal weight loss after two months. Although further studies are needed, we anticipate that targeting epigenetic regulators or use of conventional therapies in combination with MR would further potentiate this effect. Conclusions: MLLr leukemic cells have an increased dependency on S-adenosylmethionine and therefore show increased vulnerability to methionine depletion. Limiting methionine availability, either by enzymatic methionine depletion or dietary restriction could provide a novel therapeutic option for this patient group, particularly when combined with other therapies. The availability of an FDA approved methionine-free formula facilitates rapid translation to clinical practice, particularly in infants. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Activating transcription factor 4 (ATF4) is a stress-induced transcription factor that is frequently upregulated in cancer cells. ATF4 controls the expression of a wide range of adaptive genes that allow cells to endure periods of stress, such as hypoxia or amino acid limitation. However, under persistent stress conditions, ATF4 promotes the induction of apoptosis. Recent advances point to a role for post-translational modifications (PTMs) and epigenetic mechanisms in balancing these pro- and anti-survival effects of ATF4. We review here how PTMs and epigenetic modifiers associated with ATF4 may be exploited by cancer cells to cope with cellular stress conditions that are intrinsically associated with tumor growth. Identification of mechanisms that modulate ATF4-mediated transcription and its effects on cellular metabolism may uncover new targets for cancer treatment.

Contains fulltext : 202708.pdf (Publisher’s version ) (Open Access) Since the identification of B-cell translocation gene 1 (BTG1) and BTG2 as antiproliferation genes more than two decades ago, their protein products have been implicated in a variety of cellular processes including cell division, DNA repair, transcriptional regulation and messenger RNA stability. In addition to affecting differentiation during development and in the adult, BTG proteins play an important role in maintaining homeostasis under conditions of cellular stress. Genomic profiling of B-cell leukemia and lymphoma has put BTG1 and BTG2 in the spotlight, since both genes are frequently deleted or mutated in these malignancies, pointing towards a role as tumor suppressors. Moreover, in solid tumors, reduced expression of BTG1 or BTG2 is often correlated with malignant cell behavior and poor treatment outcome. Recent studies have uncovered novel roles for BTG1 and BTG2 in genotoxic and integrated stress responses, as well as during hematopoiesis. This review summarizes what is currently known about the roles of BTG1 and BTG2 in these and other cellular processes. In addition, we will highlight the molecular mechanisms and biological consequences of BTG1 and BTG2 deregulation during cancer progression and elaborate on the potential clinical implications of these findings.

Invadosomes are actin-rich membrane protrusions that degrade the extracellular matrix to drive tumor cell invasion. Key players in invadosome formation are c-Src and Rho family GTPases. Invadosomes can reassemble into circular rosette-like superstructures, but the underlying signaling mechanisms remain obscure. Here we show that Src-induced invadosomes in human melanoma cells (A375M and MDA-MB-435) undergo rapid remodeling into dynamic extracellular matrix-degrading rosettes by distinct G protein-coupled receptor agonists, notably lysophosphatidic acid (LPA; acting through the LPA1 receptor) and endothelin. Agonist-induced rosette formation is blocked by pertussis toxin, dependent on PI3K activity and accompanied by localized production of phosphatidylinositol 3,4,5-trisphosphate, whereas MAPK and Ca(2+) signaling are dispensable. Using FRET-based biosensors, we show that LPA and endothelin transiently activate Cdc42 through Gi, concurrent with a biphasic decrease in Rac activity and differential effects on RhoA. Cdc42 activity is essential for rosette formation, whereas G12/13-mediated RhoA-ROCK signaling suppresses the remodeling process. Our results reveal a Gi-mediated Cdc42 signaling axis by which G protein-coupled receptors trigger invadosome remodeling, the degree of which is dictated by the Cdc42-RhoA activity balance.

Contains fulltext : 172024.pdf (Publisher’s version ) (Closed access) Transient receptor potential (TRP) channels comprise a family of cation channels implicated in a variety of cellular processes, including proliferation, cell migration and cell survival. As a consequence, members of this ion family play prominent roles during embryonic development, tissue maintenance and cancer progression. Although most TRP channels are non-selective, many cellular responses, mediated by TRP channels, appear to be calcium-dependent. In addition, there is mounting evidence for channel-independent roles for TRP channels. In this review, we will discuss how both these channel-dependent and -independent mechanisms affect cellular programs essential during embryonic development, and how perturbations in these pathways contribute to a variety of pathologies. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen.

Contains fulltext : 168248.pdf (Publisher’s version ) (Open Access) Cancer cells are frequently exposed to physiological stress conditions such as hypoxia and nutrient limitation. Escape from stress-induced apoptosis is one of the mechanisms used by malignant cells to survive unfavorable conditions. B-cell Translocation Gene 1 (BTG1) is a tumor suppressor that is frequently deleted in acute lymphoblastic leukemia and recurrently mutated in diffuse large B cell lymphoma. Moreover, low BTG1 expression levels have been linked to poor outcome in several solid tumors. How loss of BTG1 function contributes to tumor progression is not well understood. Here, using Btg1 knockout mice, we demonstrate that loss of Btg1 provides a survival advantage to primary mouse embryonic fibroblasts (MEFs) under stress conditions. This pro-survival effect involves regulation of Activating Transcription Factor 4 (ATF4), a key mediator of cellular stress responses. We show that BTG1 interacts with ATF4 and positively modulates its activity by recruiting the protein arginine methyl transferase PRMT1 to methylate ATF4 on arginine residue 239. We further extend these findings to B-cell progenitors, by showing that loss of Btg1 expression enhances stress adaptation of mouse bone marrow-derived B cell progenitors. In conclusion, we have identified the BTG1/PRMT1 complex as a new modifier of ATF4 mediated stress responses. 14 p.