Search

Reverse phase protein arrays (RPPA) can assess protein expression and activation states in large numbers of samples (n > 1000) and evidence suggests feasibility in the setting of multi-institution clinical trials. Despite evidence in solid tumors, little is known about protein stability in leukemia. Proteins collected from leukemia cells in blood and bone marrow biopsies must be sufficiently stable for analysis. Using 58 leukemia samples, we initially assessed protein/phospho-protein integrity for the following preanalytical variables: 1) shipping vs local processing, 2) temperature (4 °C vs ambient temperature), 3) collection tube type (heparin vs Cell Save (CS) preservation tubes), 4) treatment effect (pre- vs post-chemotherapy) and 5) transit time. Next, we assessed 1515 samples from the Children's Oncology Group Phase 3 AML clinical trial (AAML1031, NCT01371981) for the effects of transit time and tube type. Protein expression from shipped blood samples was stable if processed in ≤72 h. While protein expression in pre-chemotherapy samples was stable in both heparin and CS tubes, post-chemotherapy samples were stable in only CS tubes. RPPA protein extremes is a successful quality control measure to identify and exclude poor quality samples. These data demonstrate that a majority of shipped proteins can be accurately assessed using RPPA. SIGNIFICANCE: RPPA can assess protein abundance and activation states in large numbers of samples using small amounts of material, making this method ideal for use in multi-institution clinical trials. However, there is little known about the effect of preanalytical handling variables on protein stability and the integrity of protein concentrations after sample collection and shipping. In this study, we used RPPA to assess preanalytical variables that could potentially affect protein concentrations. We found that the preanalytical variables of shipping, transit time, and temperature had minimal effects on RPPA protein concentration distributions in peripheral blood and bone marrow, demonstrating that these preanalytical variables could be successfully managed in a multi-site clinical trial setting.

In Fig. 3c of this Letter, the the effects of CRISPR-Cas9-mediated deletion of NR3C1, TXNIP and CNR2 in patient-derived B-lineage leukaemia cells were shown. For curves depicting NR3C1 (left graph), data s for TXNIP (middle graph) were inadvertently plotted. This figure has been corrected online, and the original Fig. 3c is shown as Supplementary Information to this Amendment for transparency. The error does not affect the conclusions of the Letter. In addition, Source Data files have been added for the Figs. 1-4 and Extended Data Figs. 1-10 of the original Letter.

BACKGROUND: Inotuzumab ozogamicin, an anti-CD22 monoclonal antibody bound to a toxin, calicheamicin, has shown single-agent activity in relapsed or refractory acute lymphoblastic leukaemia. We aimed to assess the activity and safety of inotuzumab ozogamicin in combination with low-intensity chemotherapy in older patients with acute lymphoblastic leukaemia. METHODS: We did a single-arm, phase 2 study at the MD Anderson Cancer Center (Houston, TX, USA). Eligible patients were aged 60 years or older and had newly diagnosed, Philadelphia chromosome-negative, acute lymphoblastic leukaemia, and an Eastern Cooperative Oncology Group performance status of 3 or lower. The induction chemotherapy regimen used was mini-hyper-CVD (a lower intensity version of the conventional hyper-CVAD). Odd-numbered cycles (1,3, 5, and 7) comprised intravenous cyclophosphamide (150 mg/m2 every 12 h on days 1-3) and oral or intravenous dexamethasone (20 mg per day on days 1-4 and days 11-14); no anthracycline was administered. Intravenous vincristine (2 mg flat dose) was given on days 1 and 8. Even-numbered cycles comprised intravenous methotrexate (250 mg/m2 on day 1) and intravenous cytarabine (0·5 g/m2 given every 12 h on days 2 and 3). Intravenous inotuzumab ozogamicin was given on day 3 of the first four cycles at the dose of 1·3-1·8 mg/m2 at cycle 1, followed by 1·0 -1·3 mg/m2 in subsequent cycles. Maintenance therapy with dose-reduced POMP (purinethol [6-mercaptopurine], oncovin [vincristine sulfate], methotrexate, and prednisone) was given for 3 years. The primary endpoint of this study was progression-free survival at 2 years. Analyses were by intention to treat. The study is ongoing, recruiting patients for an approved expansion phase with a modified treatment plan by protocol amendment. The trial is registered with ClinicalTrials.gov, number NCT01371630. FINDINGS: Between Nov 12, 2011, and April 22, 2017, 52 patients with a median age of 68 years (IQR 64-72) were enrolled. With a median follow-up of 29 months (IQR 13-48), 2-year progression-free survival was 59% (95% CI 43-72). The most frequent grade 3-4 adverse events were prolonged thrombocytopenia (42 [81%] patients), infections during induction (27 [52%]) and consolidation chemotherapy (36 [69%]), hyperglycaemia (28 [54%]), hypokalaemia (16 [31%]), increased aminotransferases (ten [19%]), hyperbilirubinaemia (nine [17%]), and haemorrhage (seven [15%]). Veno-occlusive disease occurred in four (8%) patients. Six (12%) patients died from adverse events that were deemed treatment related (five [10%] from sepsis and one [2%] from veno-occlusive disease). INTERPRETATION: Inotuzumab ozogamicin plus mini-hyper-CVD chemotherapy is a safe and active first-line therapy option in older patients with newly diagnosed acute lymphoblastic leukaemia and could represent a new therapy for this population. Randomised, phase 3 trials to evaluate the efficacy of this combination compared with the current standard of care in this setting, combination chemotherapy without inotuzumab ozogamicin, are warranted. FUNDING: MD Anderson Cancer Center.

B-lymphoid transcription factors, such as PAX5 and IKZF1, are critical for early B-cell development, yet lesions of the genes encoding these transcription factors occur in over 80% of cases of pre-B-cell acute lymphoblastic leukaemia (ALL). The importance of these lesions in ALL has, until now, remained unclear. Here, by combining studies using chromatin immunoprecipitation with sequencing and RNA sequencing, we identify a novel B-lymphoid program for transcriptional repression of glucose and energy supply. Our metabolic analyses revealed that PAX5 and IKZF1 enforce a state of chronic energy deprivation, resulting in constitutive activation of the energy-stress sensor AMPK. Dominant-negative mutants of PAX5 and IKZF1, however, relieved this glucose and energy restriction. In a transgenic pre-B ALL mouse model, the heterozygous deletion of Pax5 increased glucose uptake and ATP levels by more than 25-fold. Reconstitution of PAX5 and IKZF1 in samples from patients with pre-B ALL restored a non-permissive state and induced energy crisis and cell death. A CRISPR/Cas9-based screen of PAX5 and IKZF1 transcriptional targets identified the products of NR3C1 (encoding the glucocorticoid receptor), TXNIP (encoding a glucose-feedback sensor) and CNR2 (encoding a cannabinoid receptor) as central effectors of B-lymphoid restriction of glucose and energy supply. Notably, transport-independent lipophilic methyl-conjugates of pyruvate and tricarboxylic acid cycle metabolites bypassed the gatekeeper function of PAX5 and IKZF1 and readily enabled leukaemic transformation. Conversely, pharmacological TXNIP and CNR2 agonists and a small-molecule AMPK inhibitor strongly synergized with glucocorticoids, identifying TXNIP, CNR2 and AMPK as potential therapeutic targets. Furthermore, our results provide a mechanistic explanation for the empirical finding that glucocorticoids are effective in the treatment of B-lymphoid but not myeloid malignancies. Thus, B-lymphoid transcription factors function as metabolic gatekeepers by limiting the amount of cellular ATP to levels that are insufficient for malignant transformation.

Current standard of care for patients with pediatric acute lymphoblastic leukemia (ALL) is mainly effective, with high remission rates after treatment. However, the genetic perturbations that give rise to this disease remain largely undefined, limiting the ability to address resistant tumors or develop less toxic targeted therapies. Here, we report the use of next-generation sequencing to interrogate the genetic and pathogenic mechanisms of 240 pediatric ALL cases with their matched remission samples. Commonly mutated genes fell into several categories, including RAS/receptor tyrosine kinases, epigenetic regulators, transcription factors involved in lineage commitment, and the p53/cell-cycle pathway. Unique recurrent mutational hotspots were observed in epigenetic regulators CREBBP (R1446C/H), WHSC1 (E1099K), and the tyrosine kinase FLT3 (K663R, N676K). The mutant WHSC1 was established as a gain-of-function oncogene, while the epigenetic regulator ARID1A and transcription factor CTCF were functionally identified as potential tumor suppressors. Analysis of 28 diagnosis/relapse trio patients plus 10 relapse cases revealed four evolutionary paths and uncovered the ordering of acquisition of mutations in these patients. This study provides a detailed mutational portrait of pediatric ALL and gives insights into the molecular pathogenesis of this disease. Cancer Res; 77(2); 390-400. ©2016 AACR.

Chromosomal rearrangements are initiating events in acute lymphoblastic leukaemia (ALL). Here using RNA sequencing of 560 ALL cases, we identify rearrangements between MEF2D (myocyte enhancer factor 2D) and five genes (BCL9, CSF1R, DAZAP1, HNRNPUL1 and SS18) in 22 B progenitor ALL (B-ALL) cases with a distinct gene expression profile, the most common of which is MEF2D-BCL9. Examination of an extended cohort of 1,164 B-ALL cases identified 30 cases with MEF2D rearrangements, which include an additional fusion partner, FOXJ2; thus, MEF2D-rearranged cases comprise 5.3% of cases lacking recurring alterations. MEF2D-rearranged ALL is characterized by a distinct immunophenotype, DNA copy number alterations at the rearrangement sites, older diagnosis age and poor outcome. The rearrangements result in enhanced MEF2D transcriptional activity, lymphoid transformation, activation of HDAC9 expression and sensitive to histone deacetylase inhibitor treatment. Thus, MEF2D-rearranged ALL represents a distinct form of high-risk leukaemia, for which new therapeutic approaches should be considered.

mTOR activation leads to enhanced survival signaling in acute myeloid leukemia (AML) cells. The active-site mTOR inhibitors (asTORi) represent a promising new approach to targeting mTOR in AKT/mTOR signaling. MLN0128 is an orally-administered, second-generation asTORi, currently in clinical development. We examined the anti-leukemic effects and the mechanisms of action of MLN0128 in AML cell lines and primary samples, with a particular focus on its effect in AML stem/progenitor cells. MLN0128 inhibited cell proliferation and induced apoptosis in AML by attenuating the activity of mTOR complex 1 and 2. Using time-of-flight mass cytometry, we demonstrated that MLN0128 selectively targeted and functionally inhibited AML stem/progenitor cells with high AKT/mTOR signaling activity. Using the reverse-phase protein array technique, we measured expression and phosphorylation changes in response to MLN0128 in 151 proteins from 24 primary AML samples and identified several pro-survival pathways that antagonize MLN0128-induced cellular stress. A combined blockade of AKT/mTOR signaling and these pro-survival pathways facilitated AML cell killing. Our findings provide a rationale for the clinical use of MLN0128 to target AML and AML stem/progenitor cells, and support the use of combinatorial multi-targeted approaches in AML therapy.

Several studies reported improved outcomes of adolescents and young adults (AYA) with acute lymphoblastic leukemia (ALL) treated with pediatric-based ALL regimens. This prompted the prospective investigation of a pediatric Augmented Berlin-Frankfurt-Münster (ABFM) regimen, and its comparison with hyper-fractionated cyclophosphamide, vincristine, Adriamycin, and dexamethasone (hyper-CVAD) in AYA patients. One hundred and six AYA patients (median age 22 years) with Philadelphia chromosome- (Ph) negative ALL received ABFM from October 2006 through March 2014. Their outcome was compared to 102 AYA patients (median age 27 years), treated with hyper-CVAD at our institution. The complete remission (CR) rate was 93% with ABFM and 98% with hyper-CVAD. The 5-year complete remission duration (CRD) were 53 and 55%, respectively (P = 0.98). The 5-year overall survival (OS) rates were 60 and 60%, respectively. The MRD status on Day 29 and Day 84 of therapy was predictive of long-term outcomes on both ABFM and hyper-CVAD. Severe regimen toxicities with ABFM included hepatotoxicity in 41%, pancreatitis in 11%, osteonecrosis in 9%, and thrombosis in 19%. Myelosuppression-associated complications were most significant with hyper-CVAD. In summary, ABFM and hyper-CVAD resulted in similar efficacy outcomes, but were associated with different toxicity profiles, asparaginase-related with ABFM and myelosuppression-related with hyper-CVAD. Am. J. Hematol. 91:819-823, 2016. © 2016 Wiley Periodicals, Inc.

Acute Myeloid Leukemia (AML) is a fatal hematological cancer. The genetic abnormalities underlying AML are extremely heterogeneous among patients, making prognosis and treatment selection very difficult. While clinical proteomics data has the potential to improve prognosis accuracy, thus far, the quantitative means to do so have yet to be developed. Here we report the results and insights gained from the DREAM 9 Acute Myeloid Prediction Outcome Prediction Challenge (AML-OPC), a crowdsourcing effort designed to promote the development of quantitative methods for AML prognosis prediction. We identify the most accurate and robust models in predicting patient response to therapy, remission duration, and overall survival. We further investigate patient response to therapy, a clinically actionable prediction, and find that patients that are classified as resistant to therapy are harder to predict than responsive patients across the 31 models submitted to the challenge. The top two performing models, which held a high sensitivity to these patients, substantially utilized the proteomics data to make predictions. Using these models, we also identify which signaling proteins were useful in predicting patient therapeutic response.

More than 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large, publicly available repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment, called the Public Repository of Xenografts (PRoXe). PRoXe includes all de-identified information relevant to the primary specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs that mimic human randomized clinical trials can characterize drug efficacy and generate transcriptional, functional, and proteomic biomarkers in both treatment-naive and relapsed/refractory disease.

Phosphatase and tensin homolog (PTEN) is a negative regulator of the phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) signaling pathway and a potent tumor suppressor in many types of cancer. To test a tumor suppressive role for PTEN in pre-B acute lymphoblastic leukemia (ALL), we induced Cre-mediated deletion of Pten in mouse models of pre-B ALL. In contrast to its role as a tumor suppressor in other cancers, loss of one or both alleles of Pten caused rapid cell death of pre-B ALL cells and was sufficient to clear transplant recipient mice of leukemia. Small-molecule inhibition of PTEN in human pre-B ALL cells resulted in hyperactivation of AKT, activation of the p53 tumor suppressor cell cycle checkpoint and cell death. Loss of PTEN function in pre-B ALL cells was functionally equivalent to acute activation of autoreactive pre-B cell receptor signaling, which engaged a deletional checkpoint for the removal of autoreactive B cells. We propose that targeted inhibition of PTEN and hyperactivation of AKT triggers a checkpoint for the elimination of autoreactive B cells and represents a new strategy to overcome drug resistance in human ALL.

Chromosomal rearrangements are a hallmark of acute lymphoblastic leukemia (ALL) and are important ALL initiating events. We describe four different rearrangements of the erythropoietin receptor gene EPOR in Philadelphia chromosome-like (Ph-like) ALL. All of these rearrangements result in truncation of the cytoplasmic tail of EPOR at residues similar to those mutated in primary familial congenital polycythemia, with preservation of the proximal tyrosine essential for receptor activation and loss of distal regulatory residues. This resulted in deregulated EPOR expression, hypersensitivity to erythropoietin stimulation, and heightened JAK-STAT activation. Expression of truncated EPOR in mouse B cell progenitors induced ALL in vivo. Human leukemic cells with EPOR rearrangements were sensitive to JAK-STAT inhibition, suggesting a therapeutic option in high-risk ALL.

BackgroundPonatinib has shown efficacy in patients with refractory chronic myeloid leukaemia (CML) and in those with CML with a Thr315Ile mutation. We aimed to investigate the activity and safety of ponatinib as first-line treatment for patients with chronic-phase CML.MethodsWe did a single-arm, phase 2 trial at MD Anderson Cancer Center in Houston, TX, USA. Between May 3, 2012, and Sept 24, 2013, we enrolled patients with early (

Studying mechanisms of malignant transformation of human pre-B cells, we found that acute activation of oncogenes induced immediate cell death in the vast majority of cells. Few surviving pre-B cell clones had acquired permissiveness to oncogenic signaling by strong activation of negative feedback regulation of Erk signaling. Studying negative feedback regulation of Erk in genetic experiments at three different levels, we found that Spry2, Dusp6, and Etv5 were essential for oncogenic transformation in mouse models for pre-B acute lymphoblastic leukemia (ALL). Interestingly, a small molecule inhibitor of DUSP6 selectively induced cell death in patient-derived pre-B ALL cells and overcame conventional mechanisms of drug-resistance.

Studying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR(+) ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR(+) ALL.

BackgroundVarious trials have reported improved outcomes for adolescents and young adults (AYAs) with acute lymphoblastic leukemia (ALL) who received treatment with pediatric-based regimens. Those reports prompted the current investigation of the pediatric augmented Berlin-Frankfurt-Münster (ABFM) regimen in AYA patients. The results were compared with those from a similar population that received the hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone (hyper-CVAD) regimen.MethodsEighty-five patients ages 12 to 40 years who had Philadelphia chromosome (Ph)-negative ALL received the ABFM regimen from October 2006 through April 2012. Their outcome was compared with outcomes in 71 historic AYA patients who received hyper-CVAD from the authors' institution. Patient and disease characteristics, as well as minimal residual disease status, were analyzed for their impact on outcomes.ResultsThe complete response rate with ABFM was 94%. The 3-year complete remission duration (CRD) and overall survival (OS) rates were 70% and 74%, respectively. For patients aged ≤21 years, the 3-year CRD and OS rates were 72% and 85%, respectively; and, for patients ages 21 to 40 years, the respective rates were 69% and 60%. The initial white blood cell count was an independent predictive factor of OS and CRD. The minimal residual disease status on days 29 and 84 of therapy also were predictive of long-term outcomes. Severe regimen toxicities included transient hepatotoxicity in 35% to 39% of patients, pancreatitis in 11% of patients, osteonecrosis in 11% of patients, and thrombosis in 22% of patients. The 3-year OS rate was 74% in the ABFM group versus 71% in the hyper-CVAD group, and the corresponding 3-year CRD rate was 70% versus 66%, respectively.ConclusionsABFM was tolerable in AYA patients with ALL but was not associated with significant improvements in CRD and OS compared with hyper-CVAD.