Loss of the stress sensor GADD45A promotes stem cell activity and ferroptosis resistance in LGR4/HOXA9-dependent AML.

ABSTRACT: The overall prognosis of acute myeloid leukemia (AML) remains dismal, largely because of the inability of current therapies to kill leukemia stem cells (LSCs) with intrinsic resistance. Loss of the stress sensor growth arrest and DNA damage-inducible 45 alpha (GADD45A) is implicated in poor clinical outcomes, but its role in LSCs and AML pathogenesis is unknown. Here, we define GADD45A as a key downstream target of G protein-coupled receptor (LGR)4 pathway and discover a regulatory role for GADD45A loss in promoting leukemia-initiating activity and oxidative resistance in LGR4/HOXA9-dependent AML, a poor prognosis subset of leukemia. Knockout of GADD45A enhances AML progression in murine and patient-derived xenograft (PDX) mouse models. Deletion of GADD45A induces substantial mutations, increases LSC self-renewal and stemness in vivo, and reduces levels of reactive oxygen species (ROS), accompanied by a decreased response to ROS-associated genotoxic agents (eg, ferroptosis inducer RSL3) and acquisition of an increasingly aggressive phenotype on serial transplantation in mice. Our single-cell cellular indexing of transcriptomes and epitopes by sequencing analysis on patient-derived LSCs in PDX mice and subsequent functional studies in murine LSCs and primary AML patient cells show that loss of GADD45A is associated with resistance to ferroptosis (an iron-dependent oxidative cell death caused by ROS accumulation) through aberrant activation of antioxidant pathways related to iron and ROS detoxification, such as FTH1 and PRDX1, upregulation of which correlates with unfavorable outcomes in patients with AML. These results reveal a therapy resistance mechanism contributing to poor prognosis and support a role for GADD45A loss as a critical step for leukemia-initiating activity and as a target to overcome resistance in aggressive leukemia.

Inflammatory response to retrotransposons drives tumor drug resistance that can be prevented by reverse transcriptase inhibitors.

Activation of endogenous retrotransposons frequently occurs in cancer cells and contributes to tumor genomic instability. To test whether inhibition of retrotranspositions has an anticancer effect, we used treatment with the nucleoside reverse transcriptase inhibitor (NRTI) stavudine (STV) in mouse cancer models, MMTV-HER2/Neu and Th-MYCN, that spontaneously develop breast cancer and neuroblastoma, respectively. In both cases, STV in drinking water did not affect tumor incidence nor demonstrate direct antitumor effects. However, STV dramatically extended progression-free survival in both models following an initial complete response to chemotherapy. To approach the mechanism underlying this phenomenon, we analyzed the effect of NRTI on the selection of treatment-resistant variants in tumor cells in culture. Cultivation of mouse breast carcinoma 4T1 in the presence of STV dramatically reduced the frequency of cells capable of surviving treatment with anticancer drugs. Global transcriptome analysis demonstrated that the acquisition of drug resistance by 4T1 cells was accompanied by an increase in the constitutive activity of interferon type I and NF-κB pathways and an elevated expression of LINE-1 elements, which are known to induce inflammatory responses via their products of reverse transcription. Treatment with NRTI reduced NF-κB activity and reverted drug resistance. Furthermore, the inducible expression of LINE-1 stimulated inflammatory response and increased the frequency of drug-resistant variants in a tumor cell population. These results indicate a mechanism by which retrotransposon desilencing can stimulate tumor cell survival during treatment and suggest reverse transcriptase inhibition as a potential therapeutic approach for targeting the development of drug-resistant cancers.

Phase 1 study of high-dose DFMO, celecoxib, cyclophosphamide and topotecan for patients with relapsed neuroblastoma: a New Approaches to Neuroblastoma Therapy trial.

BACKGROUND: MYC genes regulate ornithine decarboxylase (Odc) to increase intratumoral polyamines. We conducted a Phase I trial [NCT02030964] to determine the maximum tolerated dose (MTD) of DFMO, an Odc inhibitor, with celecoxib, cyclophosphamide and topotecan. METHODS: Patients 2-30 years of age with relapsed/refractory high-risk neuroblastoma received oral DFMO at doses up to 9000 mg/m2/day, with celecoxib (500 mg/m2 daily), cyclophosphamide (250 mg/m2/day) and topotecan (0.75 mg/m2/day) IV for 5 days, for up to one year with G-CSF support. RESULTS: Twenty-four patients (median age, 6.8 years) received 136 courses. Slow platelet recovery with 21-day courses (dose-levels 1 and 2) led to subsequent dose-levels using 28-day courses (dose-levels 2a-4a). There were three course-1 dose-limiting toxicities (DLTs; hematologic; anorexia; transaminases), and 23 serious adverse events (78% fever-related). Five patients (21%) completed 1-year of therapy. Nine stopped for PD, 2 for DLT, 8 by choice. Best overall response included two PR and four MR. Median time-to-progression was 19.8 months, and 3 patients remained progression-free at >4 years without receiving additional therapy. The MTD of DFMO with this regimen was 6750 mg/m2/day. CONCLUSION: High-dose DFMO is tolerable when added to chemotherapy in heavily pre-treated patients. A randomized Phase 2 trial of DFMO added to chemoimmunotherapy is ongoing [NCT03794349].

Inhibition of mitochondrial translocase SLC25A5 and histone deacetylation is an effective combination therapy in neuroblastoma.

The mitochondrion is a gatekeeper of apoptotic processes, and mediates drug resistance to several chemotherapy agents used to treat cancer. Neuroblastoma is a common solid cancer in young children with poor clinical outcomes following conventional chemotherapy. We sought druggable mitochondrial protein targets in neuroblastoma cells. Among mitochondria-associated gene targets, we found that high expression of the mitochondrial adenine nucleotide translocase 2 (SLC25A5/ANT2), was a strong predictor of poor neuroblastoma patient prognosis and contributed to a more malignant phenotype in pre-clinical models. Inhibiting this transporter with PENAO reduced cell viability in a panel of neuroblastoma cell lines in a TP53-status-dependant manner. We identified the histone deacetylase inhibitor, suberanilohydroxamic acid (SAHA), as the most effective drug in clinical use against mutant TP53 neuroblastoma cells. SAHA and PENAO synergistically reduced cell viability, and induced apoptosis, in neuroblastoma cells independent of TP53-status. The SAHA and PENAO drug combination significantly delayed tumour progression in pre-clinical neuroblastoma mouse models, suggesting that these clinically advanced inhibitors may be effective in treating the disease.

Hopes, concerns, satisfaction and regret in a precision medicine trial for childhood cancer: a mixed-methods study of parent and patient perspectives.

BACKGROUND: Paediatric precision oncology aims to match therapeutic agents to driver gene targets. We investigated whether parents and patients regret participation in precision medicine trials, particularly when their hopes are unfulfilled. METHODS: Parents and adolescent patients completed questionnaires at trial enrolment (T0) and after receiving results (T1). Parents opted-in to an interview at T1. Bereaved parents completed a questionnaire 6-months post-bereavement (T1B). We analysed quantitative data with R and qualitative data thematically with NVivo, before integrating all data for interpretation. RESULTS: 182 parents and 23 patients completed T0; 108/182 parents and 8/23 patients completed T1; 27/98 bereaved parents completed T1B; and 45/108 parents were interviewed. At enrolment, participants held concurrent hopes that precision medicine would benefit future children and their child. Participants expressed concern regarding wait-times for receipt of results. Most participants found the trial beneficial and not burdensome, including bereaved parents. Participants reported high trial satisfaction (median scores: parents: 93/100; patients: 80/100). Participants expressed few regrets (parent median scores: parents: 10/100; bereaved parents: 15/100; patient regret: 2/8 expressed minimal regret). CONCLUSIONS: Even when trial outcomes did not match their hopes, parents and patients rarely regretted participating in a childhood cancer precision medicine trial. These data are critical for integrating participants' views into future precision medicine delivery.

Mitotic Dysregulation at Tumor Initiation Creates a Therapeutic Vulnerability to Combination Anti-Mitotic and Pro-Apoptotic Agents for MYCN-Driven Neuroblastoma.

MYCN amplification occurs in approximately 20-30% of neuroblastoma patients and correlates with poor prognosis. The TH-MYCN transgenic mouse model mimics the development of human high-risk neuroblastoma and provides strong evidence for the oncogenic function of MYCN. In this study, we identified mitotic dysregulation as a hallmark of tumor initiation in the pre-cancerous ganglia from TH-MYCN mice that persists through tumor progression. Single-cell quantitative-PCR of coeliac ganglia from 10-day-old TH-MYCN mice revealed overexpression of mitotic genes in a subpopulation of premalignant neuroblasts at a level similar to single cells derived from established tumors. Prophylactic treatment using antimitotic agents barasertib and vincristine significantly delayed the onset of tumor formation, reduced pre-malignant neuroblast hyperplasia, and prolonged survival in TH-MYCN mice. Analysis of human neuroblastoma tumor cohorts showed a strong correlation between dysregulated mitosis and features of MYCN amplification, such as MYC(N) transcriptional activity, poor overall survival, and other clinical predictors of aggressive disease. To explore the therapeutic potential of targeting mitotic dysregulation, we showed that genetic and chemical inhibition of mitosis led to selective cell death in neuroblastoma cell lines with MYCN over-expression. Moreover, combination therapy with antimitotic compounds and BCL2 inhibitors exploited mitotic stress induced by antimitotics and was synergistically toxic to neuroblastoma cell lines. These results collectively suggest that mitotic dysregulation is a key component of tumorigenesis in early neuroblasts, which can be inhibited by the combination of antimitotic compounds and pro-apoptotic compounds in MYCN-driven neuroblastoma.

Whole-genome sequencing facilitates patient-specific quantitative PCR-based minimal residual disease monitoring in acute lymphoblastic leukaemia, neuroblastoma and Ewing sarcoma.

BACKGROUND: Minimal residual disease (MRD) measurement is a cornerstone of contemporary acute lymphoblastic leukaemia (ALL) treatment. The presence of immunoglobulin (Ig) and T cell receptor (TCR) gene recombinations in leukaemic clones allows widespread use of patient-specific, DNA-based MRD assays. In contrast, paediatric solid tumour MRD remains experimental and has focussed on generic assays targeting tumour-specific messenger RNA, methylated DNA or microRNA. METHODS: We examined the feasibility of using whole-genome sequencing (WGS) data to design tumour-specific polymerase chain reaction (PCR)-based MRD tests (WGS-MRD) in 18 children with high-risk relapsed cancer, including ALL, high-risk neuroblastoma (HR-NB) and Ewing sarcoma (EWS) (n = 6 each). RESULTS: Sensitive WGS-MRD assays were generated for each patient and allowed quantitation of 1 tumour cell per 10-4 (0.01%)-10-5 (0.001%) mononuclear cells. In ALL, WGS-MRD and Ig/TCR-MRD were highly concordant. WGS-MRD assays also showed good concordance between quantitative PCR and droplet digital PCR formats. In serial clinical samples, WGS-MRD correlated with disease course. In solid tumours, WGS-MRD assays were more sensitive than RNA-MRD assays. CONCLUSIONS: WGS facilitated the development of patient-specific MRD tests in ALL, HR-NB and EWS with potential clinical utility in monitoring treatment response. WGS data could be used to design patient-specific MRD assays in a broad range of tumours.

Targeting multidrug resistance-associated protein 1 (MRP1)-expressing cancers: Beyond pharmacological inhibition.

Resistance to chemotherapy remains one of the most significant obstacles to successful cancer treatment. While inhibiting drug efflux mediated by ATP-binding cassette (ABC) transporters is a seemingly attractive and logical approach to combat multidrug resistance (MDR), small molecule inhibition of ABC transporters has so far failed to confer clinical benefit, despite considerable efforts by medicinal chemists, biologists, and clinicians. The long-sought treatment to eradicate cancers displaying ABC transporter overexpression may therefore lie within alternative targeting strategies. When aberrantly expressed, the ABC transporter multidrug resistance-associated protein 1 (MRP1, ABCC1) confers MDR, but can also shift cellular redox balance, leaving the cell vulnerable to select agents. Here, we explore the physiological roles of MRP1, the rational for targeting this transporter in cancer, the development of small molecule MRP1 inhibitors, and the most recent developments in alternative therapeutic approaches for targeting cancers with MRP1 overexpression. We discuss approaches that extend beyond simple MRP1 inhibition by exploiting the collateral sensitivity to glutathione depletion and ferroptosis, the rationale for targeting the shared transcriptional regulators of both MRP1 and glutathione biosynthesis, advances in gene silencing, and new molecules that modulate transporter activity to the detriment of the cancer cell. These strategies illustrate promising new approaches to address multidrug resistant disease that extend beyond the simple reversal of MDR and offer exciting routes for further research.

Exploiting the reactive oxygen species imbalance in high-risk paediatric acute lymphoblastic leukaemia through auranofin.

BACKGROUND: The prognosis for high-risk childhood acute leukaemias remains dismal and established treatment protocols often cause long-term side effects in survivors. This study aims to identify more effective and safer therapeutics for these patients. METHODS: A high-throughput phenotypic screen of a library of 3707 approved drugs and pharmacologically active compounds was performed to identify compounds with selective cytotoxicity against leukaemia cells followed by further preclinical evaluation in patient-derived xenograft models. RESULTS: Auranofin, an FDA-approved agent for the treatment of rheumatoid arthritis, was identified as exerting selective anti-cancer activity against leukaemia cells, including patient-derived xenograft cells from children with high-risk ALL, versus solid tumour and non-cancerous cells. It induced apoptosis in leukaemia cells by increasing reactive oxygen species (ROS) and potentiated the activity of the chemotherapeutic cytarabine against highly aggressive models of infant MLL-rearranged ALL by enhancing DNA damage accumulation. The enhanced sensitivity of leukaemia cells towards auranofin was associated with lower basal levels of the antioxidant glutathione and higher baseline ROS levels compared to solid tumour cells. CONCLUSIONS: Our study highlights auranofin as a well-tolerated drug candidate for high-risk paediatric leukaemias that warrants further preclinical investigation for application in high-risk paediatric and adult acute leukaemias.

Potent antileukemic activity of curaxin CBL0137 against MLL-rearranged leukemia.

Around 10% of acute leukemias harbor a rearrangement of the MLL/KMT2A gene, and the presence of this translocation results in a highly aggressive, therapy-resistant leukemia subtype with survival rates below 50%. There is a high unmet need to identify safer and more potent therapies for MLL-rearranged (MLL-r) leukemia that can be combined with established chemotherapeutics to decrease treatment-related toxicities. The curaxin, CBL0137, has demonstrated nongenotoxic anticancer and chemopotentiating effects in a number of preclinical cancer models and is currently in adult Phase I clinical trials for solid tumors and hematological malignancies. The aim of our study was to investigate whether CBL0137 has potential as a therapeutic and chemopotentiating compound in MLL-r leukemia through a comprehensive analysis of its efficacy in preclinical models of the disease. CBL0137 decreased the viability of a panel of MLL-r leukemia cell lines (n = 12) and xenograft cells derived from patients with MLL-r acute lymphoblastic leukemia (ALL, n = 3) in vitro with submicromolar IC50s. The small molecule drug was well-tolerated in vivo and significantly reduced leukemia burden in a subcutaneous MV4;11 MLL-r acute myeloid leukemia model and in patient-derived xenograft models of MLL-r ALL (n = 5). The in vivo efficacy of standard of care drugs used in remission induction for pediatric ALL was also potentiated by CBL0137. CBL0137 exerted its anticancer effect by trapping Facilitator of Chromatin Transcription (FACT) into chromatin, activating the p53 pathway and inducing an Interferon response. Our findings support further preclinical evaluation of CBL0137 as a new approach for the treatment of MLL-r leukemia.

ABCC4/MRP4 contributes to the aggressiveness of Myc-associated epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is a complex disease comprising discrete histological and molecular subtypes, for which survival rates remain unacceptably low. Tailored approaches for this deadly heterogeneous disease are urgently needed. Efflux pumps belonging to the ATP-binding cassette (ABC) family of transporters are known for roles in both drug resistance and cancer biology and are also highly targetable. Here we have investigated the association of ABCC4/MRP4 expression to clinical outcome and its biological function in endometrioid and serous tumors, common histological subtypes of EOC. We found high expression of ABCC4/MRP4, previously shown to be directly regulated by c-Myc/N-Myc, was associated with poor prognosis in endometrioid EOC (P = .001) as well as in a subset of serous EOC with a "high-MYCN" profile (C5/proliferative; P = .019). Transient siRNA-mediated suppression of MRP4 in EOC cells led to reduced growth, migration and invasion, with the effects being most pronounced in endometrioid and C5-like serous cells compared to non-C5 serous EOC cells. Sustained knockdown of MRP4 also sensitized endometrioid cells to MRP4 substrate drugs. Furthermore, suppression of MRP4 decreased the growth of patient-derived EOC cells in vivo. Together, our findings provide the first evidence that MRP4 plays an important role in the biology of Myc-associated ovarian tumors and highlight this transporter as a potential therapeutic target for EOC.

Accelerating development of high-risk neuroblastoma patient-derived xenograft models for preclinical testing and personalised therapy.

BACKGROUND: Predictive preclinical models play an important role in the assessment of new treatment strategies and as avatar models for personalised medicine; however, reliable and timely model generation is challenging. We investigated the feasibility of establishing patient-derived xenograft (PDX) models of high-risk neuroblastoma from a range of tumour-bearing patient materials and assessed approaches to improve engraftment efficiency. METHODS: PDX model development was attempted in NSG mice by using tumour materials from 12 patients, including primary and metastatic solid tumour samples, bone marrow, pleural fluid and residual cells from cytogenetic analysis. Subcutaneous, intramuscular and orthotopic engraftment were directly compared for three patients. RESULTS: PDX models were established for 44% (4/9) of patients at diagnosis and 100% (5/5) at relapse. In one case, attempted engraftment from pleural fluid resulted in an EBV-associated atypical lymphoid proliferation. Xenogeneic graft versus host disease was observed with attempted engraftment from lymph node and bone marrow tumour samples but could be prevented by T-cell depletion. Orthotopic engraftment was more efficient than subcutaneous or intramuscular engraftment. CONCLUSIONS: High-risk neuroblastoma PDX models can be reliably established from diverse sample types. Orthotopic implantation allows more rapid model development, increasing the likelihood of developing an avatar model within a clinically useful timeframe.

Optimization of a clofarabine-based drug combination regimen for the preclinical evaluation of pediatric acute lymphoblastic leukemia.

BACKGROUND: The aim of this study was to improve the predictive power of patient-derived xenografts (PDXs, also known as mouse avatars) to more accurately reflect outcomes of clofarabine-based treatment in pediatric acute lymphoblastic leukemia (ALL) patients. PROCEDURE: Pharmacokinetic (PK) studies were conducted using clofarabine at 3.5 to 15 mg/kg in mice. PDXs were established from relapsed/refractory ALL patients who exhibited good or poor responses to clofarabine. PDX engraftment and response to clofarabine (either as a single agent or in combinations) were assessed based on stringent objective response measures modeled after the clinical setting. RESULTS: In naïve immune-deficient NSG mice, we determined that a clofarabine dose of 3.5 mg/kg resulted in systemic exposures equivalent to those achieved in pediatric ALL patients treated with clofarabine-based regimens. This dose was markedly lower than the doses of clofarabine used in previously reported preclinical studies (typically 30-60 mg/kg) and, when scheduled consistent with the clinical regimen (daily × 5), resulted in 34-fold lower clofarabine exposures. Using a well-tolerated clofarabine/etoposide/cyclophosphamide combination regimen, we then found that the responses of PDXs better reflected the clinical responses of the patients from whom the PDXs were derived. CONCLUSIONS: This study has identified an in vivo clofarabine treatment regimen that reflects the clinical responses of relapsed/refractory pediatric ALL patients. This regimen could be used prospectively to identify patients who might benefit from clofarabine-based treatment. Our findings are an important step toward individualizing prospective patient selection for the use of clofarabine in relapsed/refractory pediatric ALL patients and highlight the need for detailed PK evaluation in murine PDX models.

Brief Report: Potent clinical and radiological response to larotrectinib in TRK fusion-driven high-grade glioma.

Genes encoding TRK are oncogenic drivers in multiple tumour types including infantile fibrosarcoma, papillary thyroid cancer and high-grade gliomas (HGG). TRK fusions have a critical role in tumourigenesis in 40% of infant HGG. Here we report the first case of a TRK fusion-driven HGG treated with larotrectinib-the first selective pan-TRK inhibitor in clinical development. This 3-year-old girl had failed multiple therapies including chemotherapy and radiotherapy. Tumour profiling confirmed an ETV6-NTRK3 fusion. Treatment with larotrectinib led to rapid clinical improvement with near total resolution of primary and metastatic lesions on MRI imaging. This is the first report of a TRK fusion glioma successfully treated with a TRK inhibitor.

A risk score including microdeletions improves relapse prediction for standard and medium risk precursor B-cell acute lymphoblastic leukaemia in children.

To prevent relapse, high risk paediatric acute lymphoblastic leukaemia (ALL) is treated very intensively. However, most patients who eventually relapse have standard or medium risk ALL with low minimal residual disease (MRD) levels. We analysed recurrent microdeletions and other clinical prognostic factors in a cohort of 475 uniformly treated non-high risk precursor B-cell ALL patients with the aim of better predicting relapse and refining risk stratification. Lower relapse-free survival at 7 years (RFS) was associated with IKZF1 intragenic deletions (P < 0·0001); P2RY8-CRLF2 gene fusion (P < 0·0004); Day 33 MRD>5 × 10-5 (P < 0·0001) and High National Cancer Institute (NCI) risk (P < 0·0001). We created a predictive model based on a risk score (RS) for deletions, MRD and NCI risk, extending from an RS of 0 (RS0) for patients with no unfavourable factors to RS2 +  for patients with 2 or 3 high risk factors. RS0, RS1, and RS2 +  groups had RFS of 93%, 78% and 49%, respectively, and overall survival (OS) of 99%, 91% and 71%. The RS provided greater discrimination than MRD-based risk stratification into standard (89% RFS, 96% OS) and medium risk groups (79% RFS, 91% OS). We conclude that this RS may enable better early therapeutic stratification and thus improve cure rates for childhood ALL.

Recommendations for the standardization of bone marrow disease assessment and reporting in children with neuroblastoma on behalf of the International Neuroblastoma Response Criteria Bone Marrow Working Group.

BACKGROUND: The current study was conducted to expedite international standardized reporting of bone marrow disease in children with neuroblastoma and to improve equivalence of care. METHODS: A multidisciplinary International Neuroblastoma Response Criteria Bone Marrow Working Group was convened by the US National Cancer Institute in January 2012 with representation from Europe, North America, and Australia. Practical transferable recommendations to standardize the reporting of bone marrow disease were developed. RESULTS: To the authors' knowledge, the current study is the first to comprehensively present consensus criteria for the collection, analysis, and reporting of the percentage area of bone marrow parenchyma occupied by tumor cells in trephine-biopsies. The quantitative analysis of neuroblastoma content in bone marrow aspirates by immunocytology and reverse transcriptase-quantitative polymerase chain reaction are revised. The inclusion of paired-like homeobox 2b (PHOX2B) for immunohistochemistry and reverse transcriptase-quantitative polymerase chain reaction is recommended. Recommendations for recording bone marrow response are provided. The authors endorse the quantitative assessment of neuroblastoma cell content in bilateral core needle biopsies-trephines and aspirates in all children with neuroblastoma, with the exception of infants, in whom the evaluation of aspirates alone is advised. It is interesting to note that 5% disease is accepted as an internationally achievable level for disease assessment. CONCLUSIONS: The quantitative assessment of neuroblastoma cells is recommended to provide data from which evidence-based numerical criteria for the reporting of bone marrow response can be realized. This is particularly important in the minimal disease setting and when neuroblastoma detection in bone marrow is intermittent, where clinical impact has yet to be validated. The wide adoption of these harmonized criteria will enhance the ability to compare outcomes from different trials and facilitate collaborative trial design. Cancer 2017;123:1095-1105. © 2016 American Cancer Society.

ABC transporters as mediators of drug resistance and contributors to cancer cell biology.

The extrusion of anticancer drugs by members of the ATP-binding cassette (ABC) transporter family is one of the most widely recognized mechanisms of multidrug resistance, and can be considered a hijacking of their normal roles in the transport of xenobiotics, metabolites and signaling molecules across cell membranes. While roles in cancer multidrug resistance have been clearly demonstrated for P-glycoprotein (P-gp), Breast Cancer Resistance Protein (BCRP) and Multidrug Resistance Protein 1 (MRP1), direct evidence for a role in multidrug resistance in vivo is lacking for other family members. A less well understood but emerging theme is the drug efflux-independent contributions of ABC transporters to cancer biology, supported by a growing body of evidence that their loss or inhibition impacts on the malignant potential of cancer cells in vitro and in vivo. As with multidrug resistance, these contributions likely represent a hijacking of normal ABC transporter functions in the efflux of endogenous metabolites and signaling molecules, however they may expand the clinical relevance of ABC transporters beyond P-gp, BCRP and MRP1. This review summarizes established and emerging roles for ABC transporters in cancer, with a focus on neuroblastoma and ovarian cancer, and considers approaches to validate and better understand these roles.

GPR84 sustains aberrant ß-catenin signaling in leukemic stem cells for maintenance of MLL leukemogenesis.

ß-catenin is required for establishment of leukemic stem cells (LSCs) in acute myeloid leukemia (AML). Targeted inhibition of ß-catenin signaling has been hampered by the lack of pathway components amenable to pharmacologic manipulation. Here we identified a novel ß-catenin regulator, GPR84, a member of the G protein-coupled receptor family that represents a highly tractable class of drug targets. High GPR84 expression levels were confirmed in human and mouse AML LSCs compared with hematopoietic stem cells (HSCs). Suppression of GPR84 significantly inhibited cell growth by inducing G1-phase cell-cycle arrest in pre-LSCs, reduced LSC frequency, and impaired reconstitution of stem cell-derived mixed-lineage leukemia (MLL) AML, which represents an aggressive and drug-resistant subtype of AML. The GPR84-deficient phenotype in established AML could be rescued by expression of constitutively active ß-catenin. Furthermore, GPR84 conferred a growth advantage to Hoxa9/Meis1a-transduced stem cells. Microarray analysis demonstrated that GPR84 significantly upregulated a small set of MLL-fusion targets and ß-catenin coeffectors, and downregulated a hematopoietic cell-cycle inhibitor. Altogether, our data reveal a previously unrecognized role of GPR84 in maintaining fully developed AML by sustaining aberrant ß-catenin signaling in LSCs, and suggest that targeting the oncogenic GPR84/ß-catenin signaling axis may represent a novel therapeutic strategy for AML.

Persistent MRD before and after allogeneic BMT predicts relapse in children with acute lymphoblastic leukaemia.

Minimal residual disease (MRD) during early chemotherapy is a powerful predictor of relapse in acute lymphoblastic leukaemia (ALL) and is used in children to determine eligibility for allogeneic haematopoietic stem cell transplantation (HSCT) in first (CR1) or later complete remission (CR2/CR3). Variables affecting HSCT outcome were analysed in 81 children from the ANZCHOG ALL8 trial. The major cause of treatment failure was relapse, with a cumulative incidence of relapse at 5 years (CIR) of 32% and treatment-related mortality of 8%. Leukaemia-free survival (LFS) and overall survival (OS) were similar for HSCT in CR1 (LFS 62%, OS 83%, n = 41) or CR2/CR3 (LFS 60%, OS 72%, n = 40). Patients achieving bone marrow MRD negativity pre-HSCT had better outcomes (LFS 83%, OS 92%) than those with persistent MRD pre-HSCT (LFS 41%, OS 64%, P < 0·0001) or post-HSCT (LFS 35%, OS 55%, P < 0·0001). Patients with B-other ALL had more relapses (CIR 50%, LFS 41%) than T-ALL and the main precursor-B subtypes including BCR-ABL1, KMT2A (MLL), ETV6-RUNX1 (TEL-AML1) and hyperdiploidy >50. A Cox multivariate regression model for LFS retained both B-other ALL subtype (hazard ratio 4·1, P = 0·0062) and MRD persistence post-HSCT (hazard ratio 3·9, P = 0·0070) as independent adverse prognostic variables. Persistent MRD could be used to direct post-HSCT therapy.