A B S T R A C T PurposeYounger patients with acute myeloid leukemia (AML) harboring NPM1 mutations without FLT3-internal tandem duplications (ITDs; NPM1-positive/FLT3-ITD-negative genotype) are classified as better risk; however, it remains uncertain whether this favorable classification can be applied to older patients with AML with this genotype. Therefore, we examined the impact of age on the prognostic significance of NPM1-positive/FLT3-ITD-negative status in older patients with AML. Patients and MethodsPatients with AML age Ն 55 years treated with intensive chemotherapy as part of Southwest Oncology Gorup (SWOG) and UK National Cancer Research Institute/Medical Research Council (NCRI/MRC) trials were evaluated. A comprehensive analysis first examined 156 patients treated in SWOG trials. Validation analyses then examined 1,258 patients treated in MRC/NCRI trials. Univariable and multivariable analyses were used to determine the impact of age on the prognostic significance of NPM1 mutations, FLT3-ITDs, and the NPM1-positive/FLT3-ITD-negative genotype. ResultsPatients with AML age 55 to 65 years with NPM1-positive/FLT3-ITD-negative genotype treated in SWOG trials had a significantly improved 2-year overall survival (OS) as compared with those without this genotype (70% v 32%; P Ͻ .001). Moreover, patients age 55 to 65 years with NPM1-positive/FLT3-ITD-negative genotype had a significantly improved 2-year OS as compared with those age Ͼ 65 years with this genotype (70% v 27%; P Ͻ .001); any potential survival benefit of this genotype in patients age Ͼ 65 years was marginal (27% v 16%; P ϭ .33). In multivariable analysis, NPM1-positive/FLT3-ITD-negative genotype remained independently associated with an improved OS in patients age 55 to 65 years (P ϭ .002) but not in those age Ͼ 65 years (P ϭ .82). These results were confirmed in validation analyses examining the NCRI/MRC patients. ConclusionNPM1-positive/FLT3-ITD-negative genotype remains a relatively favorable prognostic factor for patients with AML age 55 to 65 years but not in those age Ͼ 65 years.

The pH-responsive poly(amidoamine)s (PAAs) have been previously described. Whereas ISA23 enhances transfection in vitro and ISA1 promotes the cytosolic delivery of the non-permeant toxins this process shows poor efficiency. The aim of this study was to prepare and evaluate PAA conjugates containing the membrane disrupting peptide melittin (MLT). It was hypothesised that PAA conjugation would reduce the haemolytic activity of MLT at pH 7.4, however, upon delivery to tumours by the EPR effect, the polymer would uncoil in an acidic environment exposing MLT and allowing it to interact with membranes. PAA-MLT conjugates were prepared using MLT as a comonomer together with bis-acryloylpiperazine, 2-methylpiperazine and bis-hydroxyethylethylenediamine (ISA1-like), or bis-acrylamidoacetic acid and 2-methylpiperazine (ISA23-like). The melittin content of the conjugates was 6-19% (w/w). Although ISA1-MLT improved gelonin delivery compared to the parent polymer ISA1 (␣ 13-fold increase) and showed pH-dependent haemolytic activity at a polymer concentration of 0.05 mg/ml, this conjugate also displayed high haemolytic activity at pH 7.4. In contrast, ISA23-MLT like the parent compound ISA23 did not deliver gelonin. However, this conjugate could have potential as a novel polymeric anticancer conjugate due to its lack of haemolytic activity at pH 7.4 and retention of cytotoxicity.

Although the prognostic impact of mutations of FLT3 and NPM1 have been extensively studied in younger patients with acute myeloid leukaemia, less is known in older patients whether treated intensively or non-intensively, or in the context of existing prognostic scores. In 1312 patients 16 and 21%, respectively had an FLT3 and NPM1 mutation. An FLT3 mutation did not affect remission rate in intensively or non-intensively treated patients but was associated with an inferior survival. All patients with an NPM1c mutation had a significantly higher remission rate irrespective of treatment approach but survival was not improved, overall, or in any genotype except as in younger patients, in the FLT3 WT NPM1c mutant subgroup. When incorporated into an established multi-parameter prognostic risk score, the molecular information provided additional prognostic definition in 11% of patients.

Summary: The poly(amidoamine)s (PAAs) ISA 1 and ISA 23 display pH‐dependent conformational change and pH‐dependent membrane perturbation. These properties confer potential for use as endosomolytic polymers for intracytoplasmic delivery of toxins and genes. Both polymers are relatively non‐toxic, and moreover ISA 23 has the beneficial property in vivo, of being non hepatotropic when administered intravenously. Although ISA 23 and ISA 1 demonstrate ability to transfect cells, ISA 1 is also able to promote intracellular delivery of non‐permeant toxins. The aim of this study was to synthesise random and block copolymers of ISA 1 and ISA 23 and investigate whether these second generation hybrids would allow optimisation of PAA biological characteristics. Random and block copolymers of ISA 1 and ISA 23 were synthesised by hydrogen transfer polyaddition to generate a library of PAAs with an ISA 23:ISA 1 molar ratios of 2:1 to 4:1. The resultant polymers have a pI slightly below 7.4 and a $\overline M _{\rm w}$ of 19 900–49 000 g/mol and a $\overline M _{\rm n}$ of 13 100–24 100 g/mol. Whereas none of the random or block copolymers were haemolytic at pH 7.4 all demonstrated pH‐dependent membrane activity. At pH 5.5 they caused 50–60% haemoglobin (Hb) release over 1 h. This was slightly less than that seen for ISA 23 (80% Hb release). None of the copolymers were cytotoxic against B16F10 cells during a 72 h incubation (IC50 > 2 mg/ml; MTT assay). The ability of the random and block copolymer PAAs to deliver the toxin gelonin was also examined, but only ISA 1 and the block copolymer B2 (ISA 23:ISA 1 at a 2:1 molar ratio) were able to promote intracellular delivery, as measured by cytotoxic activity. It would be interesting to study the body distribution of B2 and determine whether this toxin‐delivering PAA is able to escape liver capture. magnified image

) is a selective cyclin-dependent kinase (CDK) inhibitor. In this study, we evaluated its effects on primary acute myeloid leukemia (AML) samples (n ¼ 87). In vitro exposure to SNS-032 for 48 h resulted in a mean LD 50 of 139±203 nM; Cytarabine (Ara-C) was more than 35 times less potent in the same cohort. SNS-032-induced a dose-dependent increase in annexin V staining and caspase-3 activation. At the molecular level, SNS-032 induced a marked dephosphorylation of serine 2 and 5 of RNA polymerase (RNA Pol) II and inhibited the expression of CDK2 and CDK9 and dephosphorylated CDK7. Furthermore, the combination of SNS-032 and Ara-C showed remarkable synergy that was associated with reduced mRNA levels of the antiapoptotic genes XIAP, BCL2 and MCL1.In conclusion, SNS-032 is effective as a single agent and in combination with Ara-C in primary AML blasts. Treatment with Ara-C alone significantly induced the transcription of the antiapoptotic genes BCL2 and XIAP. In contrast, the combination of SNS-032 and Ara-C suppressed the transcription of BCL2, XIAP and MCL1. Therefore, the combination of SNS-032 and Ara-C may increase the sensitivity of AML cells to the cytotoxic effects of Ara-C by inhibiting the transcription of antiapoptotic genes.

HighlightsGanetespib is a highly potent HSP90 inhibitor in primary AML blasts.Apoptotic induction is co-ordinate with suppression of pro-survival protein AKT.Synergistic interaction with AraC suppressing pro-survival targets HSP70 and AKT.Provides strong rationale for further clinical assessment of ganetespib in AML.

SummaryHeat shock protein 90 (HSP90; HSP90AA1) is a molecular chaperone involved in signalling pathways for cell proliferation, survival, and cellular adaptation. Inhibitors of HSP90 are being examined as anti-cancer agents, but the critical molecular mechanism(s) of their activity remains unresolved. HSP90 inhibition potentially facilitates the simultaneous targeting of multiple molecules within tumour cells and represents an attractive therapeutic proposition. Here, we investigated HSP90 as a molecular target for acute myeloid leukaemia (AML) using the novel HSP90 inhibitor NVP-AUY922-AG. NVP-AUY922-AG induced dosedependent killing in myeloid cell lines and primary AML blasts. In primary blasts, cell death in response to NVP-AUY922-AG was seen at concentrations almost 2 logs lower than cytarabine (Ara-C) (50% lethal dose = 0Á12 l mol/l AE 0Á28). NVP-AUY922-AG was significantly less toxic to normal bone marrow (P = 0Á02). In vitro response to NVP-AUY922-AG did not correlate with response to Ara-C (r 2 = 0Á0006).NVP-AUY922-AG was highly synergistic with Ara-C in cell lines and in 20/25 of the primary samples tested. NVP-AUY922-AG induced increases in HSP70 expression and depletion of total AKT, IKKa and IKKb in cell lines and primary blasts. This study shows that the novel HSP90 inhibitor NVP-AUY922-AG has significant single agent activity in AML cells and is synergistic with Ara-C.

Background There is conflicting data on the effect of the addition of ATRA to chemotherapy in AML. Two large randomised trials showed no benefit (Estey et al Blood 1999; 93, 2478 (n=215); Burnett et al. Blood 2010, 115: 9482 (n=1075)), or benefit which was limited to patients with an NPM1 mutation (n=14) when given in combination with ICE (Idarubicin/Ara-C/Etoposide) (Schlenk et al, Leukemia 2004, 18; 1798 (n=242)) but not with DA alone. In an effort to prospectively clarify if this is predictive treatment for NPM1+ patients and whether the effect was etoposide dependent, we randomised 616 patients to DA vs ADE and ATRA vs no ATRA in a 2x2 factorial design. Methods Between August 2010 and May 2012, 616 patients were randomised. The median age was 67(53-82) years: 75% had de novo, 16% had secondary, and 8% had high risk MDS (marrow blasts 10-19%): 4%, 75% and 21% had favourable, intermediate or poor risk cytogenetics: ITD and NPM1 data was available on 422 and 404 patients, with mutation rates of 19% and 24%. A total of 56 patients (14% of those with data) were molecularly good risk (NPM1 mutant, ITD wild type). By Wheatley risk score, 24%, 40% and 36% had good, standard or poor risk disease. The demographic, cytogenetic, molecular and allocated treatments were balanced between the arms. Follow-up is complete to 1st January 2013 (median follow-up 18.7 months) Patients were given Daunorubicin 50mg/m2 days 1-3 + Ara-C 100mg/m2 bid days 1-10 (course 1) or days 1-8 (course 2). Those allocated ATRA were treated at 45mg/m2/day for 60 days; Etoposide in the ADE arm was given at 100mg/m2/day on days 1-5 of courses 1 and 2 of chemotherapy. Results The overall response rate (ORR) was 69% (CR 53%+ CRi 16%) and survival at 2 years was 35%. The ORR was not different between DA: 68% (CR 53%, CRi 15%) and ADE: 70% (CR 53%, CRi 17%), odds ratio (OR) 0.92 (0.65-1.30) p=0.6, although remission rates were non-significantly lower in patients given ATRA (ORR 66% (CR 53%, CRi 13%) vs 73% (CR 54%, CRi 19%), OR 1.39 (0.98-1.95) p=0.06) with significantly higher 30-day (16% vs 8%, p=0.005) and 60-day mortality (20% vs 12%, p=0.005). There were no differences in early mortality between ADE and DA arms. At 2 years, neither survival nor RFS differed between the arms: (ADE vs DA OS: 33% vs 36%, HR 1.07 (0.86-1.32) p=0.6; RFS: 23% vs 36% HR 1.15 (0.88-1.49) p=0.3; ATRA vs Not OS: 35% vs 35% HR 1.13 (0.91-1.40) p=0.3; RFS: 31% vs 30% HR 0.93 (0.71-1.20) p=0.6). Overall there was no interaction between the two treatments (OS, test for heterogeneity p=0.12). In an analysis stratified by Etoposide and by NPM1/ITD risk group there was no significant heterogeneity of the effect of ATRA (p=0.1, Figure). Importantly, when looked at by the underlying chemotherapy, no beneficial effect of ATRA in NPM1 mutant/ITD WT patients appeared for patients receiving ADE (p=1.0 for heterogeneity). Conclusions Neither the addition of Etoposide nor ATRA improves outcomes in this group of patients, with ATRA being associated with significantly greater early mortality. Importantly, an analysis by NPM1/FLT3 genotype fails to reproduce the large benefits seen by Schlenk et al in ATRA treated patients with an NPM1 mutant/ITD wild type genotype, either overall or for patients treated with ADE, thus failing to substantiate a benefit for ATRA in either context. Based upon the results of AML16, neither Etoposide nor ATRA improve outcomes for older patients given intensive DA chemotherapy. Acknowledgments This study received research support from Cancer Research UK, and the Cardiff Experimental Cardiff Medicine Centre. Disclosures: No relevant conflicts of interest to declare.