Summary In acute myeloid leukemia (AML), the cell of origin, nature and biological consequences of initiating lesions and order of subsequent mutations remain poorly understood, as AML is typically diagnosed without observation of a pre-leukemic phase. Here, highly purified hematopoietic stem cells (HSC), progenitor and mature cell fractions from the blood of AML patients were found to contain recurrent DNMT3a mutations (DNMT3amut) at high allele frequency, but without coincident NPM1 mutations (NPM1c) present in AML blasts. DNMT3amut-bearing HSC exhibited multilineage repopulation advantage over non-mutated HSC in xenografts, establishing their identity as pre-leukemic-HSC (preL-HSC). preL-HSC were found in remission samples indicating that they survive chemotherapy. Thus DNMT3amut arises early in AML evolution, likely in HSC, leading to a clonally expanded pool of preL-HSC from which AML evolves. Our findings provide a paradigm for the detection and treatment of pre-leukemic clones before the acquisition of additional genetic lesions engenders greater therapeutic resistance.

Refractoriness to induction chemotherapy and relapse after achievement of remission are the main obstacles to cure in acute myeloid leukaemia (AML). After standard induction chemotherapy, patients are assigned to different post-remission strategies on the basis of cytogenetic and molecular abnormalities that broadly define adverse, intermediate and favourable risk categories. However, some patients do not respond to induction therapy and another subset will eventually relapse despite the lack of adverse risk factors. There is an urgent need for better biomarkers to identify these high-risk patients before starting induction chemotherapy, to enable testing of alternative induction strategies in clinical trials. The high rate of relapse in AML has been attributed to the persistence of leukaemia stem cells (LSCs), which possess a number of stem cell properties, including quiescence, that are linked to therapy resistance. Here, to develop predictive and/or prognostic biomarkers related to stemness, we generated a list of genes that are differentially expressed between 138 LSC and 89 LSC cell fractions from 78 AML patients validated by xenotransplantation. To extract the core transcriptional components of stemness relevant to clinical outcomes, we performed sparse regression analysis of LSC gene expression against survival in a large training cohort, generating a 17-gene LSC score (LSC17). The LSC17 score was highly prognostic in five independent cohorts comprising patients of diverse AML subtypes (n = 908) and contributed greatly to accurate prediction of initial therapy resistance. Patients with high LSC17 scores had poor outcomes with current treatments including allogeneic stem cell transplantation. The LSC17 score provides clinicians with a rapid and powerful tool to identify AML patients who do not benefit from standard therapy and who should be enrolled in trials evaluating novel upfront or post-remission strategies.

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In a randomized trial of therapy for FMS-like tyrosine kinase-3 (FLT3) mutant acute myeloid leukemia in first relapse, 224 patients received chemotherapy alone or followed by 80 mg of the FLT3 inhibitor lestaurtinib twice daily. Endpoints included complete remission or complete remission with incomplete platelet recovery (CR/CRp), overall survival, safety, and tolerability. Correlative studies included pharmacokinetics and analysis of in vivo FLT3 inhibition. There were 29 patients with CR/CRp in the lestaurtinib arm and 23 in the control arm (26% vs 21%; P = .35), and no difference in overall survival between the 2 arms. There was evidence of toxicity in the lestaurtinib-treated patients, particularly those with plasma levels in excess of 20 μM. In the lestaurtinib arm, FLT3 inhibition was highly correlated with remission rate, but target inhibition on day 15 was achieved in only 58% of patients receiving lestaurtinib. Given that such a small proportion of patients on this trial achieved sustained FLT3 inhibition in vivo, any conclusions regarding the efficacy of combining FLT3 inhibition with chemotherapy are limited. Overall, lestaurtinib treatment after chemotherapy did not increase response rates or prolong survival of patients with FLT3 mutant acute myeloid leukemia in first relapse. This study is registered at www.clinicaltrials.gov as #NCT00079482.

The mTOR complex 2 (mTORC2) containing mTOR and rictor is thought to be rapamycin insensitive and was recently shown to regulate the prosurvival kinase AKT by phosphorylation on Ser473. We investigated the molecular effects of mTOR inhibition by the rapamycin derivatives ( IntroductionThe mammalian target of rapamycin (mTOR) pathway regulates cell growth, proliferation, and survival. 1 mTOR, the central component of this pathway, partitions between 2 scaffold proteins, raptor and rictor. Upon activation, the rapamycin-sensitive raptor/mTOR protein complex (mTORC1) increases mRNA translation via activation of p70S6-kinase and inhibition of eIF4E-binding protein 4EPB1. 2 The rictor/mTOR protein complex (mTORC2) was discovered only recently, is thought to be rapamycin insensitive, and phosphorylates AKT in the hydrophobic Ser473 site. It is therefore essential for AKT activity. 3 Despite activity in model systems, the clinical antitumor activity of rapamycin derivatives in patients has been modest, 1,4 and only a fraction of patients responds (reviewed in Thomas 5 ). This has been attributed to the unanticipated ability of rapamycin to increase AKT activity via release of feedback inhibition of growth signaling pathways, both in cell systems and in tumor biopsies from patients. 6 However, in certain cell types, prolonged inhibition of mTOR by rapamycin may impair mTORC2 assembly and hence AKT activation. 7 In this study, we investigated the molecular consequences of mTOR inhibition in leukemic cells, both in vitro and in a clinical trial in vivo. Our results demonstrate that rapamycin derivatives suppress assembly of mTORC2, resulting in marked inhibition of AKT signaling. We propose that rapamycin-induced functional blockade of AKT in leukemic cells may define a subset of hematologic malignancies that is likely to respond favorably to mTOR inhibition, and that inhibition of AKT signaling may serve as a valuable biomarker of mTOR inhibition in vivo. Materials and methodsAcute myeloid leukemia (AML) cell lines were cultured under standard conditions 8 with rapamycin derivatives CCI-779 and RAD001. Bone marrow or peripheral blood samples for the in vitro studies were obtained from patients with newly diagnosed or recurrent acute myeloid leukemia (AML) after informed consent. Peripheral blood samples were obtained from relapsed or refractory patients with hematologic malignancies treated with CCI-779 (temsirolimus; Wyeth Pharmaceuticals, Pearl River, NY) or RAD001 (everolimus; Novartis Pharmaceuticals, East Hanover, NJ) 9 after obtaining written informed consent. Approval was obtained from the M. D. Anderson Cancer Center institutional review board for these studies. Clinical characteristics of patients are summarized in Table S1 (available on the Blood website; see the Supplemental Materials link at the top of the online article). Expression of total and phosphorylated AKT (Ser473), p70S6K (Thr389), 4EBP1 (Thr70), FoxO1 (Ser256), and PTEN was detected by Western blot analysis as previously reported. 7 mTOR was immun...

Purpose RG7112 is a small-molecule MDM2 antagonist. MDM2 is a negative regulator of the tumor suppressor p53 and frequently overexpressed in leukemias. Thus, a Phase I study of RG7112 in patients with hematologic malignancies was conducted. Experimental Design Primary study objectives included determination of the dose and safety profile of RG7112. Secondary objectives included evaluation of pharmacokinetics, pharmacodynamics, such as TP53-mutation status and MDM2 expression, and preliminary clinical activity. Patients were divided into 2 cohorts: Stratum A (relapsed/refractory AML (except APL), ALL, and CML) and Stratum B (relapsed/refractory CLL/sCLL). Some Stratum A patients were treated at the MTD to assess clinical activity. Results RG7112 was administered to 116 patients (96 patients in Stratum A and 20 patients in Stratum B). All patients experienced at least 1 adverse event, and 3 DLTs were reported. PK analysis indicated that twice-daily dosing enhanced daily exposure. Anti-leukemia activity was observed in the 30 patients with AML assessed at the MTD included 5 patients who met IWG criteria for response. Exploratory analysis revealed TP53 mutations in 14% of Stratum A patients and in 40% of Stratum B patients. Two patients with TP53 mutations exhibited clinical activity. p53 target genes were induced only in TP53 wild-type leukemic cells. Baseline expression levels of MDM2 correlated positively with clinical response. Conclusions RG7112 demonstrated clinical activity against relapsed/refractory AML and CLL/sCLL. MDM2 inhibition resulted in p53 stabilization and transcriptional activation of p53-target genes. We provide proof-of-concept that MDM2 inhibition restores p53 function and generates clinical responses in hematologic malignancies.

Purpose: Everolimus (RAD001, Novartis), an oral derivative of rapamycin, inhibits the mammalian target of rapamycin (mTOR), which regulates many aspects of cell growth and division.

Summary Background Guadecitabine (SGI-110) is a novel hypomethylating dinucleotide of decitabine (DAC) and deoxyguanosine that is resistant to degradation by cytidine deaminase. Methods This is a first-in-human pharmacokinetic (PK)- and pharmacodynamic (PD)-guided Phase 1 dose-escalation study in adults with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Patients with MDS or AML refractory to, or relapsed after, standard treatment were randomly assigned to one of two regimens of subcutaneous (SC) guadecitabine: Daily×5 or Once Weekly for three weeks. Stratification was based on disease (MDS vs. AML). Treatment assignment was not blinded. A Twice Weekly for three weeks regimen was later added to the study. All regimens were given in 28-day cycles. The primary objective was the safety profile of all regimens and the recommended dose and schedule for phase 2 by either maximum tolerated dose (MTD) or biologically effective dose (BED). All patients who received at least one treatment were included in the analyses. Enrollment is complete and all patients have finished treatment. This study is registered with ClinicalTrials.gov, number NCT01261312. Findings 93 patients were treated (74 AML and 19 MDS): 44 on Daily×5 (3–125 mg/m2/d), 34 on Once Weekly (6–125 mg/m2/d), and 15 on Twice Weekly (60 and 90 mg/m2/d). Guadecitabine SC produced a longer exposure window and half-life, and lower Cmax, of plasma DAC than intravenous DAC. The MTD was 90 mg/m2 in MDS on the Daily×5 regimen but was not reached in AML or on the other regimens. The most common Grade ≥3 adverse events were febrile neutropenia (38/93, 41%), pneumonia (27/93, 29%), thrombocytopenia and anemia (23/93, 25% each), and sepsis (16/93, 17%). The most common serious adverse events (SAEs) were febrile neutropenia (29/93, 31%), pneumonia (26/93, 28%), and sepsis (16/93, 17%). Potent dose-related DNA demethylation occurred on the daily regimen, reaching a plateau at 60 mg/m2 Daily×5 (designated as BED). Responses were seen in heavily pretreated patients including six responders (two complete response [CR], two CR with incomplete blood count recovery [CRi], one CR with incomplete platelet recovery [CRp], and one partial response [PR]) in AML patients and two marrow complete response (mCR) in MDS patients. Responders showed significantly more demethylation than non-responders. Interpretation Guadecitabine SC at 60 mg/m2 Daily×5 is well-tolerated, easily administered, and biologically and clinically active in both MDS and AML; it warrants testing in phase 2 studies.