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Internal tandem duplication of FLT3 (FLT3-ITD) is one of the most common somatic mutations in acute myeloid leukemia (AML); it causes constitutive activation of FLT3 kinase and is associated with high relapse rates and poor survival. Small-molecule inhibition of FLT3 represents an attractive therapeutic strategy for this subtype of AML, although resistance from secondary FLT3 tyrosine kinase domain (FLT3-TKD) mutations is an emerging clinical problem. CCT241736 is an orally bioavailable, selective, and potent dual inhibitor of FLT3 and Aurora kinases. FLT3-ITD+ cells with secondary FLT3-TKD mutations have high in vitro relative resistance to the FLT3 inhibitors quizartinib and sorafenib, but not to CCT241736. The mechanism of action of CCT241736 results in significant in vivo efficacy, with inhibition of tumor growth observed in efficacy studies in FLT3-ITD and FLT3-ITD-TKD human tumor xenograft models. The efficacy of CCT241736 was also confirmed in primary samples from AML patients, including those with quizartinib-resistant disease, which induces apoptosis through inhibition of both FLT3 and Aurora kinases. The unique combination of CCT241736 properties based on robust potency, dual selectivity, and significant in vivo activity indicate that CCT241736 is a bona fide clinical drug candidate for FLT3-ITD and TKD AML patients with resistance to current drugs.

Previous studies have shown that sphingosine kinase interacting protein (SKIP) inhibits sphingosine kinase (SK) function in fibroblasts. SK phosphorylates sphingosine producing the potent signaling molecule sphingosine-1-phosphate (S1P). SKIP gene (SPHKAP) expression is silenced by hypermethylation of its promoter in acute myeloid leukemia (AML). However, why SKIP activity is silenced in primary AML cells is unclear. Here, we investigated the consequences of SKIP down-regulation in AML primary cells and the effects of SKIP re-expression in leukemic cell lines. Using targeted ultra-HPLC-tandem MS (UPLC-MS/MS), we measured sphingolipids (including S1P and ceramides) in AML and control cells. Primary AML cells had significantly lower SK activity and intracellular S1P concentrations than control cells, and SKIP-transfected leukemia cell lines exhibited increased SK activity. These findings show that SKIP re-expression enhances SK activity in leukemia cells. Furthermore, other bioactive sphingolipids such as ceramide were also down-regulated in primary AML cells. Of note, SKIP re-expression in leukemia cells increased ceramide levels 2-fold, inactivated the key signaling protein extracellular signal-regulated kinase, and increased apoptosis following serum deprivation or chemotherapy. These results indicate that SKIP down-regulation in AML reduces SK activity and ceramide levels, an effect that ultimately inhibits apoptosis in leukemia cells. The findings of our study contrast with previous results indicating that SKIP inhibits SK function in fibroblasts and therefore challenge the notion that SKIP always inhibits SK activity.

The specific niche adaptations that facilitate primary disease and Acute Lymphoblastic Leukaemia (ALL) survival after induction chemotherapy remain unclear. Here, we show that Bone Marrow (BM) adipocytes dynamically evolve during ALL pathogenesis and therapy, transitioning from cellular depletion in the primary leukaemia niche to a fully reconstituted state upon remission induction. Functionally, adipocyte niches elicit a fate switch in ALL cells towards slow-proliferation and cellular quiescence, highlighting the critical contribution of the adipocyte dynamic to disease establishment and chemotherapy resistance. Mechanistically, adipocyte niche interaction targets posttranscriptional networks and suppresses protein biosynthesis in ALL cells. Treatment with general control nonderepressible 2 inhibitor (GCN2ib) alleviates adipocyte-mediated translational repression and rescues ALL cell quiescence thereby significantly reducing the cytoprotective effect of adipocytes against chemotherapy and other extrinsic stressors. These data establish how adipocyte driven restrictions of the ALL proteome benefit ALL tumours, preventing their elimination, and suggest ways to manipulate adipocyte-mediated ALL resistance., How the bone marrow microenvironment evolves during induction chemotherapy to facilitate acute lymphoblastic leukaemia (ALL) survival remains unclear. Here the authors show that adipocytes emergent during ALL therapy aid the survival of ALL cells through a restrictive effect on the ALL proteome.

Background Chimeric antigen receptor (CAR)-T cell therapy has revolutionised the treatment of relapsed or refractory B-ALL in children and young adults with unprecedented response rates. However, primary resistance and relapse are unresolved challenges that limit long term benefit in a significant proportion of patients. Death receptor mediated extrinsic apoptosis is a key component of CAR-T cytotoxicity and impairment of this system, of which CAR-T derived TRAIL (tumour necrosis factor related apoptosis inducing ligand) is a key initiator, is a principal driver of primary resistance. Arginine deprivation with the therapeutic enzyme ADI-PEG20 (pegylated arginine deiminase) sensitises cancers deficient in the enzyme argininosuccinate synthase (ASS1) to the apoptosis initiating activity of TRAIL through tumour cell surface upregulation of death receptors DR4 and DR5. Whether this effect could potentiate the TRAIL-DR activity in CAR-T therapy has not been explored. Aim We tested the hypothesis that ADI-PEG20 treatment can sensitise susceptible B-ALL to anti-CD19 CAR-T through priming of death receptor mediated apoptosis signalling. Methods The effect of ADI-PEG20 on cell survival and death receptor expression in B-ALL cell lines and primary samples was analysed by flow cytometry. Second generation anti-CD19 CAR-T cells with a CD28 costimulatory domain were generated by retroviral transduction of activated peripheral blood mononuclear cells (PBMC) from healthy donors. For CAR-T co-culture experiments, B-ALL cell lines were pre-treated with ADI-PEG20 before washing and re-suspending in arginine replete media prior to CAR-T cell addition. Results To establish potential susceptibility of B-ALL to ADI-PEG20 we measured expression of ASS1, which inversely correlates with sensitivity to the drug, using combined in situ immunohistochemistry (n=6) and RT-qPCR (n=7). ASS1 deficiency was consistently seen in this series of primary samples suggesting the potential utility of ADI-PEG20 in B-ALL, with comparable expression levels to those seen in a cohort of primary AML samples proven to be sensitive to the drug (figure 1a). Next, to examine variation in ASS1 expression between genetically defined subtypes of B-ALL we re-analysed transcriptome data from a cohort of 215 patients treated on the ECOG E2993 trial, filtered into a network of 58 genes generated according to known or predicted interaction with ASS1. We found an enrichment of Philadelphia chromosome positive (Ph+) and Philadelphia-like (Ph-L) samples in the cluster characterised by lowest ASS1 expression along with high HIF1A expression, matching a recurrent pattern reported in other ADI-PEG20 sensitive tumours. This therefore predicts that among B-ALL subtypes, Ph+ and Ph-L are likely to be most sensitive to therapeutic arginine deprivation. We then functionally confirmed, using in vitro cell line (n=3) and in vivo patient derived xenograft models of B-ALL (n=2), that ASS1 deficiency is required for ADI-PEG20 sensitivity. Using the ASS1-low, Ph-L cell line MUTZ-5, we established that ADI-PEG20 induced apoptosis accompanies cell surface upregulation of both DR4 and DR5 expression. Upregulation of DR4 was observed to follow an upwards trend after treated cells were washed and re-suspended in arginine replete media, suggesting that transient arginine starvation can commit ASS1-low B-ALL to a state of apoptotic priming (figure 1b). With confirmed engagement of arginine starvation and death receptor upregulation we tested the synergy potential of ADI-PEG20 pre-treatment of MUTZ-5 followed by CAR-T, utilising calculated combination drug indices (CDIs). Across independent PBMC donors (n=3) we observed greater potency killing of CD19 + leukaemia cells in the combination treated co-cultures when compared to the single agent treated conditions, with CDIs consistently less than 1 confirming a synergistic effect (figure 1c). Conclusion Our study proposes a synergistic interaction between the arginine depleting enzyme ADI-PEG20 and anti-CD19 CAR-T for the treatment of ASS1 deficient B-ALL, whereby priming of death receptor signalling may underlie enhanced CAR-T cytotoxicity against CD19 + tumour cells. These data support an emerging framework for CAR-T optimisation based on targeting of the death receptor mediated extrinsic apoptosis pathway and can inform future refinements in the development of cellular immunotherapy. Figure 1 Figure 1. Disclosures Bomalaski: Polaris Pharmaceuticals Inc.: Current Employment. Maher: Leucid Bio: Other: Chief Scientific Officer. Gribben: Abbvie: Honoraria; AZ: Honoraria, Research Funding; BMS: Honoraria; Gilead/Kite: Honoraria; Janssen: Honoraria, Research Funding; Morphosys: Honoraria; Novartis: Honoraria; Takeda: Honoraria; TG Therapeutis: Honoraria.

Background: The poor-risk cytogenetic subgroup of acute myeloid leukaemia (AML) includes various chromosomal aberrations and represents a heterogeneous population of patients with a dismal 10-year overall survival. While the success of genetic landscaping studies is encouraging, it is debatable whether genomics, or indeed any single-omics platform alone, is sufficient to capture the biology of a disease that continues to evade our existing treatments so effectively. Instead, we need to develop a much better understanding of the complexity of this subgroup of AMLs: the relationship and interdependencies across biochemical pathways, how these may differ between patients and their impact on the leukemia and normal stem cell compartments. To launch this process, we have completed a multi-omics profiling programme to shed new light on the genetic and biochemical features of poor-risk AML (https://poor-risk-aml.bham.ac.uk/). Aims: Application of multi-omics and integrative approaches to decipher the complexities of cytogenetically poor-risk AML Methods: Sample inclusion criteria were based on cytogenetics and availability of sufficient diagnostic bone marrow or peripheral blood material for analysis. The 50 primary AMLs included 17 cases with complex karyotype, 13 -7/del(7), 11 KMT2A rearrangements (with the exception of t(9;11)), 4 t(6;9), 3 -5/de(5), 1 del(17) and 1 inv(3). Profiles consisted of a combination of genomics (whole genome sequencing (WGS, 60X for tumour and 30X for germ-line controls), targeted sequencing of 54 myeloid loci, and total RNA-seq (100 million reads per bulk sample), mass spectrometry proteomics and phosphoproteomics (with >6,000 proteins and > 25,000 phosphorylation sites detected and quantified), mass cytometry (CyTOF, 39 markers), drug screening (ranging from 200-500 approved or investigational compounds) and the selective generation of patient-derived xenograft (PDX) models. Results: Near complete datasets have been compiled on all 50 primary AMLs, with the exception of WGS analysis where profiling was restricted to cases where corresponding germline DNA was available. Integration of WGS and RNA-seq data identified 122 genes having notable allele-specific expression (ASE) in ≥ 5 samples supported by ≥ 3 SNPs and these included the transcription factor GATA2 and the DNA topoisomerase TOP1MT. Use of RNA fusion capture tools resolved novel inter- and intra- chromosomal gene rearrangements that were confirmed by WGS. The four t(6;9)(p23;q34)/DEK-NUP214 cases, with a mean age of diagnosis of 43.5 years and all harboring FLT3-ITD mutations, arose from the most immature hematopoietic compartment (CD34+CD117+ enrichment) and demonstrated a unique transcriptomic signature, which included upregulation of FOXO3 and GRP56. Collectively, t(6;9) primary samples also showed a selective drug sensitivity to XPO1 (selinexor and eltanexor) and JAK inhibitors (ruxolitinib, tofacitinib and momelotinib) compared to other cytogenetic risk groups. On the other hand, a comparison of in vitro drug sensitivity data with genomic data of our entire cohort of patients demonstrated that TP53 wt AMLs (n=37) were more sensitive to all four MDM2 inhibitors (AMG-232, idasanutlin, SAR405838 and NVP-CGM097) compared to TP53 mutated cases (n=13). Comparisons of transcriptomics with the in vitro sensitivity to drugs included in early/late phase AML clinical trials, identified signatures of response associated with MDM2 and Aurora B kinase (AZD1152-HQPA) inhibitors. Phosphoproteomics analysis and machine learning modeling separated KMT2A rearranged leukemias into 2 discrete groups (group A: MLLT4, MLLT10 and TET1; group B with MLLT6, ELL and SEP9 fusion partners). Functionally, group A presented with elevated HOXA10 protein expression and enhanced in vitro response to genotoxic drugs and cell cycle inhibitors when compared to group B leukemia. Conclusions: Our study demonstrates the feasibility of simultaneously generating omics data from several different platforms and highlights that a combination of genetic and proteomic profiles may help to inform the choice of therapies based on the underlying biology of a patient's AML. Disclosures Wennerberg: Novartis: Research Funding; Pfizer: Honoraria. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding; Innovative Mediicines Initiative project Harmony: Research Funding.

Acute myeloid leukemia (AML) disrupts the generation of normal blood cells, predisposing patients to hemorrhage, anemia, and infections. Differentiation and proliferation of residual normal hematopoietic stem and progenitor cells (HSPCs) are impeded in AML-infiltrated bone marrow (BM). The underlying mechanisms and interactions of residual hematopoietic stem cells (HSCs) within the leukemic niche are poorly understood, especially in the human context. To mimic AML infiltration and dissect the cellular crosstalk in human BM, we established humanized ex vivo and in vivo niche models comprising AML cells, normal HSPCs, and mesenchymal stromal cells (MSCs). Both models replicated the suppression of phenotypically defined HSPC differentiation without affecting their viability. As occurs in AML patients, the majority of HSPCs were quiescent and showed enrichment of functional HSCs. HSPC suppression was largely dependent on secreted factors produced by transcriptionally remodeled MSCs. Secretome analysis and functional validation revealed MSC-derived stanniocalcin 1 (STC1) and its transcriptional regulator HIF-1α as limiting factors for HSPC proliferation. Abrogation of either STC1 or HIF-1α alleviated HSPC suppression by AML. This study provides a humanized model to study the crosstalk among HSPCs, leukemia, and their MSC niche, and a molecular mechanism whereby AML impairs normal hematopoiesis by remodeling the mesenchymal niche.

The strategy of enzymatic degradation of amino acids to deprive malignant cells of important nutrients is an established component of induction therapy of acute lymphoblastic leukemia. Here we show that acute myeloid leukemia (AML) cells from most patients with AML are deficient in a critical enzyme required for arginine synthesis, argininosuccinate synthetase-1 (ASS1). Thus, these ASS1-deficient AML cells are dependent on importing extracellular arginine. We therefore investigated the effect of plasma arginine deprivation using pegylated arginine deiminase (ADI-PEG 20) against primary AMLs in a xenograft model and in vitro. ADI-PEG 20 alone induced responses in 19 of 38 AMLs in vitro and 3 of 6 AMLs in vivo, leading to caspase activation in sensitive AMLs. ADI-PEG 20-resistant AMLs showed higher relative expression of ASS1 than sensitive AMLs. This suggests that the resistant AMLs survive by producing arginine through this metabolic pathway and ASS1 expression could be used as a biomarker for response. Sensitive AMLs showed more avid uptake of arginine from the extracellular environment consistent with their auxotrophy for arginine. The combination of ADI-PEG 20 and cytarabine chemotherapy was more effective than either treatment alone resulting in responses in 6 of 6 AMLs tested in vivo. Our data show that arginine deprivation is a reasonable strategy in AML that paves the way for clinical trials.

Proteomic and genomic integration identifies kinase and differentiation determinants of kinase inhibitor sensitivity in leukemia cells

Distinct patterns of DNA methylation characterize the epigenetic landscape of promyelocytic leukemia/retinoic acid receptor-α (PML-RARα)-associated acute promyelocytic leukemia (APL). We previously reported that the microRNAs (miRNAs) clustered on chromosome 14q32 are overexpressed only in APL. Here, using high-throughput bisulfite sequencing, we identified an APL-associated hypermethylation at the upstream differentially methylated region (DMR), which also included the site motifs for the enhancer blocking protein CCCTC-binding factor (CTCF). Comparing the profiles of diagnostic/remission paired patient samples, we show that hypermethylation was acquired in APL in a monoallelic manner. The cytosine guanine dinucleotide status of the DMR correlated with expression of the miRNAs following a characteristic position-dependent pattern. Moreover, a signature of hypermethylation was also detected in leukemic cells from an established transgenic PML-RARA APL mouse model at the orthologous region on chromosome 12, including the CTCF binding site located upstream from the mouse miRNA cluster. These results, together with the demonstration that the region does not show DNA methylation changes during myeloid differentiation, provide evidence that 14q32 hypermethylation is implicated in the pathogenesis of APL. We propose a model in which loss of imprinting at the 14q32 domain leads to overexpression of the miRNAs in APL.

Leukemia-initiating cells (LICs) in acute myeloid leukemia (AML) are believed to be restricted to the CD34+ fraction. However, one of the most frequently mutated genes in AML is nucleophosmin (NPM), and this is associated with low CD34 expression. We, therefore, investigated whether NPM-mutated AMLs have LICs restricted to the CD34+ fraction. We transplanted sorted fractions of primary NPM-mutated AML into immunodeficient mice to establish which fractions initiate leukemia. Approximately one-half of cases had LICs exclusively within the CD34− fraction, whereas the CD34+ fraction contained normal multilineage hematopoietic repopulating cells. Most of the remaining cases had LICs in both CD34+ and CD34− fractions. When samples were sorted based on CD34 and CD38 expression, multiple fractions initiated leukemia in primary and secondary recipients. The data indicate that the phenotype of LICs is more heterogeneous than previously realized and can vary even within a single sample. This feature of LICs may make them particularly difficult to eradicate using therapies targeted against surface antigens.

Inherited GH insensitivity (GHI) is usually caused by mutations in the GH receptor (GHR). Patients present with short stature associated with high GH and low IGF-I levels and may have midfacial hypoplasia (typical Laron syndrome facial features). We previously described four mildly affected GHI patients with an intronic mutation in the GHR gene (A(-1)--G(-1) substitution in intron 6), resulting in the activation of a pseudoexon (6Psi) and inclusion of 36 amino acids.The study aimed to analyze the clinical and genetic characteristics of additional GHI patients with the pseudoexon (6Psi) mutation.Auxological, biochemical, genetic, and haplotype data from seven patients with severe short stature and biochemical evidence of GHI were assessed.We assessed genotype-phenotype relationship.One patient belongs to the same extended family, previously reported. She has normal facial features, and her IGF-I levels are in the low-normal range for age. The six unrelated patients, four of whom have typical Laron syndrome facial features, have heights ranging from -3.3 to -6.0 sd and IGF-I levels that vary from normal to undetectable. We hypothesize that the marked difference in biochemical and clinical phenotypes might be caused by variations in the splicing efficiency of the pseudoexon.Activation of the pseudoexon in the GHR gene can lead to a variety of GHI phenotypes. Therefore, screening for the presence of this mutation should be performed in all GHI patients without mutations in the coding exons.

Context: Patients with panhypopituitarism have impaired quality of life (QoL) despite GH replacement. They are profoundly androgen deficient, and dehydroepiandrosterone (DHEA) has been shown to have a beneficial effect on well-being and mood in patients with adrenal failure and possibly in hypopituitarism.Objective: Our objective was to determine the effect of DHEA administration on mood in hypopituitary adults on established GH replacement with a constant serum IGF-I.Design: A double-blind, placebo-controlled trial was conducted over an initial 6 months followed by an open phase of 6 months of DHEA.Setting: The study was conducted at a tertiary referral endocrinology unit.Patients: Thirty female and 21 male hypopituitary patients enrolled. Data from 26 females and 18 males were analyzed after patient withdrawal.Interventions: DHEA (50 mg) was added to maintenance replacement including GH.Main Outcome Measures: The primary outcome objective was the effect on QoL and libido assessed by QoL assessment in GH deficiency in adults, Short Form 36, General Health Questionnaire, EuroQol, and sexual self-efficacy scale.Results: Patients had impaired QoL at baseline compared with the age-matched British population. Females showed improvement in QoL assessment in GH deficiency in adults score (−2.9 ± 2.8 DHEA vs.−0.53 ± 3 placebo; P < 0.05), in Short Form 36 social functioning (14.6 ± 23.1 DHEA vs.−4.7 ± 25 placebo; P = 0.047), and general health perception (9.6 ± 14.2 DHEA vs.−1.2 ± 11.6 placebo; P = 0.036) after 6 months of DHEA. Men showed improvement in self-esteem (−1.3 ± 1.7 DHEA vs. 0.5 ± 1.5 placebo; P = 0.03) and depression (−1.6 ± 2.2 DHEA vs. 1.2 ± 2.4 placebo, P = 0.02) domains of the General Health Questionnaire after 6 months of DHEA.Conclusions: DHEA replacement leads to modest improvement in psychological well-being in female and minor psychological improvement in male hypopituitary patients on GH replacement.

Chronic inflammation is characterised by modifications in cytokine concentrations, whereas growth is mainly dependent on the GH-IGF axis. IGF-I bioavailability is modulated by a family of IGF-binding proteins (IGFBPs). The aim of the present study was to evaluate the interactions among interleukin-1beta (IL-1beta), IL-6 and IGFBP secretion by intestinal cells to assess whether cytokines modulate IGFBP secretion, and in turn IGF-I and IGF-II bioavailability. The human colon carcinoma derived cell line Caco-2 was used as an in vitro model for its capacity to differentiate spontaneously. Experiments were carried out on day 4 (undifferentiated state) and day 14 (differentiated state) after plating. Carcinoembryonic antigen (CEA) was used as a marker of differentiation and increased in the conditioned media (CM) from days 4 to 14 (0.2+/-0.01 ng/ml per 10(5) cells vs 3.3+/-0.2 ng/ml per 10(5) cells, P

Measurement of the insulin-like growth factors (IGFs) and their binding proteins has become commonplace in the indirect assessment of the integrity of the GH axis. However, the relative effect of GH deficiency (GHD) on each component of the IGF axis and the merit of any one parameter as a diagnostic test have not been defined in a homogeneous population across all ages. We therefore measured IGF-I, IGF-II, IGF-binding protein-1 (IGFBP-1), IGFBP-2, IGFBP-3, and acid labile subunit (ALS) in 27 GHD subjects (aged 5–82 yr) from an extended kindred in Northeast Brazil with an identical GHRH receptor mutation and in 55 indigenous controls (aged 5–80 yr). The effect of GHD on the theoretical distribution of IGFs between the IGFBPs and the ternary complex was also examined. All components of the IGF axis, measured and theoretical, showed complete separation between GHD and control subjects, except IGFBP-1 and IGFBP-2 concentrations, which did not differ. The most profound effects of GHD were on total IGF-I, IGF-I in the ternary complex, and ALS. The proportion of IGF-I associated with IGFBP-3 remained constant throughout life, but was significantly lower in GHD due to an increase in IGF-I/IGFBP-2 complexes. IGF-I in the ternary complex was determined principally by concentrations of ALS in GHD and IGFBP-3 in controls, implying that ALS has greater GH dependency. In the controls, IGF-II was associated primarily with IGFBP-3 and to a lesser extent with IGFBP-2, whereas in GHD the reverse was found. There was also a dramatic decline in the proportion of free ALS in GHD adults that was not evident in controls. As diagnostic tests, IGF-I in the ternary complex and total IGF-I provided the greatest separation between GHD and controls in childhood. Similarly, in older adults the best separation was achieved with IGF-I in the ternary complex, with free ALS being optimal in younger adults. Severe GHD not only reduces the amounts of IGFs, IGFBP-3, and ALS, but also modifies the distribution of the IGFs bound to each IGFBP. Diagnostic tests used in the investigation of GHD should be tailored to the age of the individual. In particular, measurement of IGF-I in the ternary complex may prove useful in the diagnosis of GHD in children and older adults, whereas free ALS may be more relevant to younger adults.

Acute myeloid leukemia (AML) induces bone marrow (BM) failure in patients, predisposing them to life-threatening infections and bleeding. The mechanism by which AML mediates this complication is unknown but one widely accepted explanation is that AML depletes the BM of hematopoietic stem cells (HSCs) through displacement. We sought to investigate how AML affects hematopoiesis by quantifying residual normal hematopoietic subpopulations in the BM of immunodeficient mice transplanted with human AML cells with a range of genetic lesions. The numbers of normal mouse HSCs were preserved whereas normal progenitors and other downstream hematopoietic cells were reduced following transplantation of primary AMLs, findings consistent with a differentiation block at the HSC–progenitor transition, rather than displacement. Once removed from the leukemic environment, residual normal hematopoietic cells differentiated normally and outcompeted steady-state hematopoietic cells, indicating that this effect is reversible. We confirmed the clinical significance of this by ex vivo analysis of normal hematopoietic subpopulations from BM of 16 patients with AML. This analysis demonstrated that the numbers of normal CD34 + CD38 − stem-progenitor cells were similar in the BM of AML patients and controls, whereas normal CD34 + CD38 + progenitors were reduced. Residual normal CD34 + cells from patients with AML were enriched in long-term culture, initiating cells and repopulating cells compared with controls. In conclusion the data do not support the idea that BM failure in AML is due to HSC depletion. Rather, AML inhibits production of downstream hematopoietic cells by impeding differentiation at the HSC–progenitor transition.

Introduction Protein kinases play a key role in how cells respond and adapt to intra and extracellular stimuli. By the addition of phosphate groups to serine, threonine or tyrosine residues, these enzymes modify the activity and properties of the targeted proteins which in turn modulate biological processes like proliferation, differentiation and cell death. Kinase signalling pathways are deregulated in most cancer types including haematological malignancies. Indeed, the kinases FLt-3, c-Kit and JAK2 as well as the up-stream kinase signalling regulators KRAS and NRAS are among the most frequently mutated genes in acute myeloid leukaemia (AML). Consequently, protein kinases have attracted the attention of the pharmaceutical and biotechnology companies and inhibitors have been found for one fifth of human kinases. In the case of AML, midostaurin, a multi-kinase inhibitor that targets, among others, the tyrosine kinase Flt-3, has granted a breakthrough therapy designation by the FDA and several other kinase inhibitors are in clinical trials or under preclinical investigation. Molecular profiling of patient samples will play a pivotal role for the development and implementation of personalized therapies including those based on kinase inhibitors. We used a molecular profile generated by a phosphoproteomics approach to rationalize why some primary AML cells respond to treatment with different kinase inhibitors while others are resistant to the same treatments. Methods Label free phosphoproteomics based on trypsin digestion and TiO2 phosphoenrichment was used to quantify > 5,000 phosphorylation sites in mononuclear cells extracted from the peripheral blood of 36 AML patients. KSEA technology was applied to infer kinase activity from the phosphoproteomics data and DAVID software was used to determine gene ontology enrichments based on the genes that code for the proteins where the phosphorylation sites were detected. Guava EasyCyte Flow Cytometry was used to determine cell viability after the treatment of the same patient samples with different kinase inhibitors. Mass cytometry was used to measure the expression at the plasma membrane of 17 surface markers in 30 of the previously analysed AML primary samples. Results The FAB classification subdivide AML cases depending on cytomorphological features. We compared the phosphoproteomes of M1 and M4 classes that are associated with early and late states of differentiation. Based on the 150 phosphopeptides more significantly regulated between FAB-M1 and FAB-M4 groups, hierarchical clustering analysis was used to stratify AML patient samples into two subsets named M1-Like and M4-Like. Phosphoproteome reanalysis showed that the M4-Like set upregulated 1255 phosphopeptides and downregulated 446 when compared with the M1-Like set. The upregulated group comprised regulatory phosphorylation sites in several kinases including PAK1 and PCK delta. Kinase activity analysis using KSEA (Kinase Substrate Enrichment Analysis) also showed an increased activity of PAK, PKCδ and other kinases like P38 alpha in the M4-Like group. Interestingly, the PAK inhibitor PF03758309 reduced more efficiently the viability in M4-Like group than in the M1-Like group (average reduction after a 72h treatment with 1µM of 55.2% for M4-Like compared to 33.8% for M1-Like, p-value = 0.0078). This difference was not observed for other inhibitors such as those targeting CK2 or p38. CyTOF analysis showed that the M4-Like group upregulated the surface expression of several differentiation markers. Discussion Predicting the effectiveness of a drug for a particular patient is a major goal of personalized medicine. In the case of kinase inhibitors, responses may be influenced by several factors including the activity of the targeted kinase as well as the activity of other kinases that act in parallel pro-survival pathways. In this work, we have found that differentiation leads to a particular activation pattern of the signalling networks, a phenomenon that determines the response to signalling inhibitors. Conclusion We found phosphoproteomics signatures in primary AML that are associated with distinct haematopoietic differentiation stages. These signatures are in turn associated with how AML cells respond to kinase inhibitors. Disclosures Fitzgibbon: Epizyme: Research Funding; Gilead: Honoraria; Janssen: Honoraria; Celgene: Honoraria.

ObjectiveGH insensitivity (GHI) is caused in the majority of cases by impaired function of the GH receptor (GHR). All but one knownGHRmutation are in the coding sequence or the exon/intron boundaries. We identified and characterised the first intronic defect occurring in the polypyrimidine tract of theGHRin a patient with severe GHI.DesignWe investigated the effect of the novel defect on mRNA splicing using anin vitrosplicing assay and a cell transfection system.MethodsGHRwas analysed by direct sequencing. To assess the effect of the novel defect, two heterologous minigenes (wild-type and mutant L1-GHR8-L2) were generated by insertingGHRexon 8 and its flanking wild-type or mutant intronic sequences into a well-characterised splicing reporter (Adml-par L1–L2).32P-labelled pre-mRNA was generated from the two constructs and incubated in HeLa nuclear extracts or HEK293 cells.ResultsSequencing of theGHRrevealed a novel homozygous defect in the polypyrimidine tract of intron 7 (IVS7-6T>A). This base change does not involve the highly conserved splice site sequences, and is not predictedin silicoto affect GHR mRNA splicing. Nevertheless, skipping of exon 8 from the mutant L1-GHR8-L2 mRNA was clearly demonstrated in thein vitrosplicing assay and in transfected HEK293 cells.ConclusionDisruption of theGHRpolypyrimidine tract causes aberrant mRNA splicing leading to a mutant GHR protein. This is predicted to lack its transmembrane and intracellular domains and, thus, be incapable of transducing a GH signal.

Immunodeficient mice are increasingly used to assay human hematopoietic repopulating cells as well as leukemia-initiating cells. One method commonly used to isolate these rare cells is to sort cells stained with fluorochrome-conjugated antibodies into fractions, then transplant the different fractions into immunodeficient mice to test their repopulating ability. The antibodies are generally treated as being neutral in terms of their effects on the experiment. Human repopulating cells are thought to express CD34 and lack CD38. Here we present evidence that anti-CD38 antibodies have a profound inhibitory effect on engraftment of cord blood and leukemia cells. We show that this effect is Fc-mediated and can be overcome by treating mice with immunosuppressive antibodies. When this inhibitory effect is prevented, we demonstrate that the CD34+CD38+ fraction of certain acute myeloid leukemia samples contains all, or at least most, leukemia-initiating cell capacity. This study highlights the potential pitfall of antibody-mediated clearance of repopulating cells and is important for any groups working with this model. More importantly, the work suggests that there is greater variation in the phenotypes of leukemia-initiating cells than previously suggested.

CONTEXT: Non-islet cell tumor hypoglycemia (NICTH) results from the hypersecretion of pro-IGF-II by a large, usually mesenchymal tumor. Detection of pro-IGF-II in serum is a potential tumor marker in these patients. OBJECTIVE: The aim of this study was to validate a rapid and reliable method for determining serum pro-IGF-II. PATIENTS: Serum samples from 16 patients with NICTH were studied. MAIN OUTCOME MEASURE: The main outcome measure was serum concentration of pro-IGF-II determined by immunoblot analysis of pro-IGF-II and mature IGF-II after 16.5% tricine-SDS-PAGE, which was compared with pro-IGF-II measured by standard RIA after size-exclusion acid chromatography. RESULTS: The analyses of patients' sera by size-exclusion acid chromatography showed that 68 +/- 19% of IGF-II were present in the pro-IGF-II form, whereas only 18 +/- 4% corresponded to pro-IGF-II in controls. Scanning densitometry of immunoblots showed 67 +/- 16% in the bands corresponding to pro-IGF-II in patients' sera, compared with 27 +/- 9% in controls. The detection sensitivity of tricine-SDS-PAGE method was the same as for size-exclusion chromatography, but the tricine-SDS-PAGE method is quicker and requires smaller amounts of serum. CONCLUSION: Tricine-SDS-PAGE followed by IGF-II immunoblot analysis provides a rapid, reproducible, and sensitive method for the separation of serum pro-IGF-II from mature IGF-II and is a useful laboratory evaluation of patients with a clinical diagnosis of NICTH.

We have previously reported a 17.2-yr-old boy with severe growth retardation and undetectable serum levels of insulin-like growth factor I (IGF-I) due to a partial deletion of the IGF-I gene. The aim of this study was to investigate the effects of recombinant human IGF-I (rhIGF-I) therapy on the GH-IGF system of this patient to gain further insights into its growth-promoting and metabolic actions. To assess the changes in GH, IGFs, IGF-binding proteins (IGFBPs), acid-labile subunit (ALS), and insulin levels, blood samples were obtained before therapy and during the first year of treatment. Hormones were analyzed by specific RIAs. Overnight GH profiles were performed before and at 1, 6, and 12 months of therapy. Fasting ALS, IGF-II, IGFBP-3, IGFBP-2, IGFBP-1, and insulin levels before rhIGF-I treatment were 46.3 mg/L, 1044 microg/L, 5.8 mg/L, 73 ng/mL, 4.7 ng/mL, and 27.3 mU/L, respectively. IGF-II, ALS, and insulin levels were elevated, whereas IGFBP-1 and IGFBP-2 levels were decreased compared to reference values. Twenty-four hours after a single s.c. injection of rhIGF-I (40 microg/kg), the concentrations were 46 mg/L, 888 microg/L, 6.9 mg/L, 112 ng/mL, 5.0 ng/mL, and 21.0 mU/L, respectively. After a single s.c. injection of rhIGF-I of 40 or 80 microg/kg x day and modelling the data using a two-compartment model, the half-lives of elimination were 15.7 and 14.3 h, with a maximum increase in IGF-I levels to 341 and 794 microg/L around 7 h, respectively. An increase in IGFBP-3 levels was observed with both doses of rhIGF-I, with a peak values of 9 mg/L. GH profiles showed a decrease in peak amplitude from 342 to 84 mU/L at 1 month, to 67 mU/L at 6 months, and to 40 mU/L at 1 yr of therapy, with no significant changes in peak number. A significant increase in IGFBP-1 levels was observed during treatment with 80 microg/kg x day IGF-I, reflecting the inhibitory effect of rhIGF-I on insulin secretion. The clinical response to rhIGF-I therapy was an increased height velocity from 3.8 cm/yr before treatment to 6.6 cm/yr. Increased lean body mass correlated with changes in the doses of rhIGF-I and, in turn, with the biochemical changes in the GH-IGF axis. Similar to healthy individuals, this patient had normal IGFBP-3 and ALS levels, which are the major regulators of the pharmacokinetics of rhIGF-I. In summary, rhIGF-I treatment has improved linear growth and insulin sensitivity in this patient by restoring IGF-I levels and by normalizing circulating GH, IGFBP, and insulin levels.

Introduction Malignant cells require amino acids for a wide range of core functions. Amino acid deprivation using enzymatic degradation has been used to induce remission in acute lymphoblastic leukemia for decades. Amino acid deprivation may also benefit patients with acute myeloid leukemia (AML). We have previously shown that AML cells lack of argininosuccinate synthetase 1(ASS1), a key enzyme in the pathway that produces arginine (1). Here we tested the effect of an arginine depleting agent, pegylated arginine deiminase (ADI-PEG 20) on primary AML cells in a xenograft model of AML. Methods NOD/SCID/interleukin 2 gamma chain null (NSG) mice were transplanted with 6 primary AML samples. 12 weeks after transplantation of AML mice received either ADI-PEG 20, cytarabine, ADI-PEG 20 plus cytarabine or vehicle. ADI was administered weekly for 4 doses and cytarabine was given for 10 consecutive days (0.2mg per day, roughly equivalent to 40mg in humans). Five weeks after treatment started, mice were killed and the percentage of AML in the bone marrow was determined by flow cytometry. Blood was collected to quantify plasma arginine using liquid chromatography-mass spectrometry/mass spectrometry. Results Plasma arginine levels were depressed following ADI-PEG 20 administration confirming that arginine depletion was achieved in vivo. In all six experiments the combination of ADI-PEG 20 and cytarabine induced a significant reduction in levels of AML compared to control (Figure 1). Critically the combination of ADI-PEG 20 and cytarabine was significantly better than cytarabine alone in three of six experiments. Conclusion Our experiments show that arginine deprivation by ADI-PEG 20 can decrease the leukemic burden in mice transplanted with primary AML cells. The combination of ADI with cytarabine had a greater effect than cytarabine alone in half the experiments. These results provide the rationale to test ADI-PEG 20 with cytarabine in clinical trials. 1. Peter W. Szlosarek, Fiona Luong, Andrew Clear, David Taussig, Simon Joel, Maria Calaminici, Silvana Debernardi, Jude Fitzgibbon, John S. Bomalaski, Arthur E. Frankel, and Dominique Bonnet. Pegylated arginine deiminase (ADI-PEG 20) as a potential novel therapy for argininosuccinate synthetase-deficient acute myeloid leukemia. Cancer Research: April 15, 2011. AACR 102nd Annual Meeting 2011, (AACR Abstract # 467) F. M-M and L. A-M contributed equally D.B., P.W.S. and D.C.T contributed equally Disclosures: Bomalaski: Polaris Group: Employment, Equity Ownership. Szlosarek:Polaris Group: Research Funding.

Abstract 2699 Transformation from chronic myelomonocytic leukemia (CMML) to acute myeloid leukemia (AML) is associated with a poor prognosis. Clinical features have been used to score those at high risk, e.g. increased marrow blast percentage. Recently, several gene mutations have been identified including TET2, KRAS, NRAS, CBL and RUNX1, of which TET2 is the most frequent (Kohlmann et al 2010, Kosmider et al 2009). Initial descriptions of TET2 mutations in myeloid malignancy suggested that they were early mutations (Delhommeau et al 2009), however subsequent studies have shown in some patients with myeloproliferative neoplasia (MPN) they can be preceded by mutations in JAK2 (Schaub et al 2010). We report a male patient who was diagnosed with CMML aged 43 years. His marrow blast count was 6% and within 2 months he had transformed to AML (FAB M4) with marrow blasts of 50%. He was treated with daunorubicin and AraC (Barts 12 schedule) and obtained a complete morphological remission (CR). However he relapsed 88 days after CR in his skin, central nervous system and marrow. He died with conservative treatment after 6 months. Using this case as a model for transforming CMML, we aimed to identify clonal changes characteristic of the three stages of his disease, chronic leukaemia, transformation to acute leukemia and subsequent relapse. We used cytogenetic analysis, single nucleotide polymorphism (SNP) arrays (Affymetrix SNP 6.0 and 10K; 10K data previously reported by Raghavan et al 2008) to identify the clonal changes. Gene mutation analysis of genomic and cDNA was performed using PCR, cloning and Sanger and amplicon deep-sequencing for the mutational burden of TET2, CBL and KRAS (454 Life Sciences, Branford, CT). At all stages of his disease, he had a normal karyotype, a NPM1 mutation, but no mutations of FLT3, JAK2, KRAS or CBL (Table). Uniparental disomy (UPD) was demonstrated on chromosome 4q at relapse but not present at diagnosis, suggesting an associated TET2 mutation that would be heterozygous at diagnosis and homozygous at relapse. The heterozygous mutation was indentified in the CMML and at transformation, in a region adjacent to the splice site of the end of exon 7. The transcript resulted in a mis-splicing event leading to a stop codon. A second truncated mutant due to a deletion in exon 6 was found expressed at low level. Deep-sequencing of genomic DNA confirmed a deletion in exon 6 consistent with this product at 2.4% (918-fold coverage) in the CMML, and increased to 5.7% (865-fold coverage) burden at transformation. At relapse, the genomic mutation was homozygous. Expression of only the mis-spliced product was observed, with no wild type TET2, and no evidence of the second truncated product by cloning or deep-sequencing (832 reads). Homozygous TET2 mutations are associated with 4qUPD in MPN and myelodysplastic syndromes (Mohamedali et al 2009, Janowska et al 2010), but this is the first description of clonal evolution of a hetero- to homozygous TET2 mutation at disease progression. The case is consistent with the hypothesis that TET2 mutations evolve early in the pathogenesis of myeloid malignancies. However, it also shows there is considerable instability of TET2 mutations, with multiple mutations at various levels, and with mitotic recombination being a late event leading to relapse. CMML Transformed AML Relapsed AML CGN 46,XY 46,XY 46,XY SNP array Not done No somatic copy number abnormalities detected 4qUPD 88201900-qter TET2 c4340+5G>T+10_+21del het result: Cys1298X c4340+5G>T+10_+21del het result: Cys1298X c4340+5G>T+10_+21del hom result: Cys1298X c3732_3733delCT 2.4% result: Tyr1245LeufsX22 c3732_3733delCT 5.7% result: Tyr1245LeufsX22 Absent NPM1 Dup956_959TCTG het Dup956_959TCTG het Dup956_959TCTG het Disclosure: No relevant conflicts of interest to declare.