Amino Acid Deprivation Response Research Articles

Aminopeptidase N Inhibitors as Pointers for Overcoming Antitumor Treatment Resistance.

Aminopeptidase N (APN), also known as CD13 antigen or membrane alanyl aminopeptidase, belongs to the M1 family of the MA clan of zinc metallopeptidases. In cancer cells, the inhibition of aminopeptidases including APN causes the phenomenon termed the amino acid deprivation response (AADR), a stress response characterized by the upregulation of amino acid transporters and synthetic enzymes and activation of stress-related pathways such as nuclear factor kB (NFkB) and other pro-apoptotic regulators, which leads to cancer cell death by apoptosis. Recently, APN inhibition has been shown to augment DR4-induced tumor cell death and thus overcome resistance to cancer treatment with DR4-ligand TRAIL, which is available as a recombinant soluble form dulanermin. This implies that APN inhibitors could serve as potential weapons for overcoming cancer treatment resistance. In this study, a series of basically substituted acetamidophenones and the semicarbazones and thiosemicarbazones derived from them were prepared, for which APN inhibitory activity was determined. In addition, a selective anti-proliferative activity against cancer cells expressing APN was demonstrated. Our semicarbazones and thiosemicarbazones are the first compounds of these structural types of Schiff bases that were reported to inhibit not only a zinc-dependent aminopeptidase of the M1 family but also a metalloenzyme.

Impaired skeletal muscle hypertrophy signaling and amino acid deprivation response in Apoe knockout mice with an unhealthy lipoprotein distribution

This study explores if unhealthy lipoprotein distribution (LPD) impairs the anabolic and amino acid sensing responses to whey-protein feeding. Thus, if impairment of such anabolic response to protein consumption is seen by the LPD this may negatively affect the skeletal muscle mass. Muscle protein synthesis (MPS) was measured by puromycin labeling in Apolipoprotein E knockout (Apoe KO), characterized by an unhealthy LPD, and wild type mice post-absorptive at 10 and 20 weeks, and post-prandial after whey-protein feeding at 20 weeks. Hypertrophy signaling and amino acid sensing mechanisms were studied and gut microbiome diversity explored. Surprisingly, whey-protein feeding did not affect MPS. p-mTOR and p-4E-BP1 was increased 2 h after whey-protein feeding in both genotypes, but with general lower levels in Apoe KO compared to wild type. At 20 weeks of age, Apoe KO had a greater mRNA-expression for SNAT2, CD98, ATF4 and GCN2 compared to wild type. These responses were not associated with gut microbiota compositional differences. Regardless of LPD status, MPS was similar in Apoe KO and wild type. Surprisingly, whey-protein did not stimulate MPS. However, Apoe KO had lower levels of hypertrophy signaling, was amino acid deprived, and had impaired amino acid sensing mechanisms.

Loss of tRNA-modifying enzyme Elp3 activates a p53-dependent antitumor checkpoint in hematopoiesis.

The hematopoietic system is highly sensitive to perturbations in the translational machinery, of which an emerging level of regulation lies in the epitranscriptomic modification of transfer RNAs (tRNAs). Here, we interrogate the role of tRNA anticodon modifications in hematopoiesis by using mouse models of conditional inactivation of Elp3, the catalytic subunit of Elongator that modifies wobble uridine in specific tRNAs. Loss of Elp3 causes bone marrow failure by inducing death in committing progenitors and compromises the grafting activity of hematopoietic stem cells. Mechanistically, Elp3 deficiency activates a p53-dependent checkpoint in what resembles a misguided amino acid deprivation response that is accompanied by Atf4 overactivation and increased protein synthesis. While deletion of p53 rescues hematopoiesis, loss of Elp3 prompts the development of p53-mutated leukemia/lymphoma, and inactivation of p53 and Elongator cooperatively promotes tumorigenesis. Specific tRNA-modifying enzymes thus condition differentiation and antitumor fate decisions in hematopoietic stem cells and progenitors.

Brain Tumor Stem Cell Dependence on Glutaminase Reveals a Metabolic Vulnerability through the Amino Acid Deprivation Response Pathway.

Cancer cells can metabolize glutamine to replenish TCA cycle intermediates, leading to a dependence on glutaminolysis for cell survival. However, a mechanistic understanding of the role that glutamine metabolism has on the survival of glioblastoma (GBM) brain tumor stem cells (BTSC) has not yet been elucidated. Here, we report that across a panel of 19 GBM BTSC lines, inhibition of glutaminase (GLS) showed a variable response from complete blockade of cell growth to absolute resistance. Surprisingly, BTSC sensitivity to GLS inhibition was a result of reduced intracellular glutamate triggering the amino acid deprivation response (AADR) and not due to the contribution of glutaminolysis to the TCA cycle. Moreover, BTSC sensitivity to GLS inhibition negatively correlated with expression of the astrocytic glutamate transporters EAAT1 and EAAT2. Blocking glutamate transport in BTSCs with high EAAT1/EAAT2 expression rendered cells susceptible to GLS inhibition, triggering the AADR and limiting cell growth. These findings uncover a unique metabolic vulnerability in BTSCs and support the therapeutic targeting of upstream activators and downstream effectors of the AADR pathway in GBM. Moreover, they demonstrate that gene expression patterns reflecting the cellular hierarchy of the tissue of origin can alter the metabolic requirements of the cancer stem cell population. SIGNIFICANCE: Glioblastoma brain tumor stem cells with low astrocytic glutamate transporter expression are dependent on GLS to maintain intracellular glutamate to prevent the amino acid deprivation response and cell death.

Resistance to Nutrient Deprivation Via Metabolic Re-Programming As Mechanism of ‘Metastasis’ Promotion in Multiple Myeloma

Aim: Multiple Myeloma (MM) is a disease of the intra-medullary (IM) compartment but with progression myeloma cells can survive in nutrient deprived extramedullary (EM) sites. Autophagy mediates the degradation and re-cycling of intra-cellular proteins and may promote tumor resistance to metabolic stressors, including nutrient deprivation. Using the MM cell lines TK1 and TK2, contemporaneously propagated from bone marrow (IM) and peripheral blood (EM), respectively, of a MM patient we investigated the role of autophagy in resistance to glutamine (Gln) deprivation.Methods & Results:TK2 but not TK1 exhibited up-regulated autophagy under both basal and Gln deprived conditions with higher LC3BII/I turnover on immunoblotting and autophagic vacuole formation on electron microscopy following chloroquine (CQ) exposure. Under conditions of Gln deprivation, TK2 was able to proliferate until day 14, whereas TK1 stopped proliferating at day 3. This proliferative advantage under Gln deprivation was abrogated by autophagy inhibitors (CQ or 3-MA) as determined by both viable cell enumeration (p=0.008 at day 7, inhibitor vs no inhibitor) and Ki67 expression (p=0.0017) only in TK2. Modulation of Gln concentration demonstrated an inverse correlation with the viability of TK1 but not TK2 - TK1 cell death increased from 36% at Gln 8mM to 56% at Gln 0mM, irrespective of autophagy inhibition. In contrast, while Gln deprivation had no significant effect on the viability of TK2, under Gln deprivation CQ exposure induced a 2-fold increase cell death measured by annexin V/PI positivity and 2.5-fold increase of cPARP expression (p=0.0045).RNAseq and GSEA analysis showed enrichment of the Krige Amino Acid Deprivation Response (AADR) pathway (ES=0.083) for TK1 with up-regulated expression of Gln transporters (SLC7A5, SLC1A5, SLC38A5 and SLC7A11). In contrast, Glutamine Synthetase (GS) expression, which is responsible for Gln synthesis, was up-regulated in TK2 cells. Interestingly, immunoblotting analysis revealed that Gln deprivation was able to induce the expression of GS while further inducing autophagic flux (2.66 fold increase of LC3II expression at Gln=0mM) only in TK2, whereas it inhibited mTOR activity in both cell lines. Additionally, GS chemical inhibition under Gln deprivation induced cell death in TK2 but not TK1 (32.56% vs 2.5% increase, respectively) demonstrating that Gln synthesis enables TK2 to overcome Gln deprivation.Conclusion. Metabolic reprogramming mitigating against nutrient deprivation may promote a more ‘metastatic’ phenotype in advanced MM. DisclosuresSpencer:Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen-Cilag: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Research Funding; BMS: Research Funding; Takeda: Honoraria, Research Funding, Speakers Bureau; STA: Honoraria.

Statins Potentiate Aminopeptidase Inhibitor (pro)Drug Activity in Acute Myeloid Leukemia Cells

Tosedostat represents a next generation oral aminopeptidase inhibitor prodrug that has recently demonstrated promising activity in elderly patients with relapsed and refractory acute myeloid leukemia (AML). This prodrug is bio-activated to its hydrophilic active metabolite by intracellular esterases including carboxylesterase 1 (CES1) hence enhancing its cellular retention and promoting targeting of multiple aminopeptidases.Aminopeptidase inhibition provokes an amino acid deprivation response, inhibition of mTOR activity and blockade of protein synthesis. The fact that CES1 also has an important physiological function in cholesterol metabolism (i.e. conversion of cholesteryl-esters to cholesterol), and the notion that AML cells display an aberrant cholesterol metabolism, prompted us to explore whether or not statins, as inhibitors of the mevalonate-cholesterol biosynthesis pathway, can potentiate the cytotoxic activity of aminopeptidase inhibitor prodrugs.Here, we discovered that the antitumor activity of CHR2863, a close structural analogue of Tosedostat, is markedly potentiated by non-toxic and clinically achievable concentrations of statins. These findings were supported by the following lines of experimental evidence; (i) A strong synergy in cell growth inhibition (combination indices < 0.1) of CHR2863 with various statins (simvastatin, fluvastatin, lovastatin and pravastatin) was demonstrated for U937 AML cells and U937 sublines displaying acquired resistance to CHR2863. (ii) This potent synergy between CHR2863 and simvastatin was also observed with a spectrum of human AML cell lines including THP1, MV4-11, and KG1, but not with acute lymphocytic leukemia (CCRF-CEM) or solid tumor cell lines (MCF7 breast cancer, KB nasopharyngeal carcinoma and SW1573 non-small cell lung cancer). (iii) The synergistic effects with non-toxic concentrations of statins were specific for aminopeptidase inhibitors, either prodrugs (CHR2863) or direct inhibitors (e.g. Bestatin), but not for other unrelated cytotoxic agents, including daunorubicin and an esterase-dependent prodrug of a histone deacetylase inhibitor. (iv) The synergy of CHR2863 with statins was also corroborated by enhanced apoptosis induction and cell cycle arrest which increased the sub-G1 fraction. (v) Statin potentiation of CHR2863 activity was abrogated by co-administration with mevalonate or farnesylpyrophosphate, and partly by geranylgeranylpyrophosphate, suggesting the involvement of protein prenylation. Consistenly, the farnesyltransferase inhibitor FTI-277 also synergized with CHR2863 activity. (vi) Mechanistically, non-toxic concentrations of simvastatin impaired Rheb prenylation which is required for lysosomal membrane association and activation of mTOR. Hence, we propose that the dual inhibitory effect of impaired Rheb prenylation by statins and CHR2863-induced mTOR inhibition achieves a potent synergistic inhibition on human AML cells. (viii) Finally, retrospective analysis in a clinical trial of relapsed and refractory elderly AML patients treated with combination chemotherapy including Tosedostat (Cortes et al, Lancet Oncol 2013), revealed a clinical benefit for patients who used a statin. AML patients on statins (n=8) and Tosedostat (switching from 240 to 120 mg/m2) had a 50% probability of 6 months survival as compared to 3 months survival for patients not on statins (n= 27). The two-year overall survival (20%) was not impacted by statin administration.Collectively, these novel findings uncover a potent therapeutic combination of statins and aminopeptidase inhibitors for the treatment of AML. This drug combination warrants further clinical evaluation, particularly in the context of elderly patients using statins for other comorbidities. DisclosuresCloos:Takeda: Research Funding; Merus: Research Funding; Novartis: Research Funding; Helsinn: Research Funding; Johnson&Johnson: Research Funding. Wang:CTI BioPharma: Employment, Equity Ownership. Singer:CTI BioPharma: Employment, Other: Stock options. Cortes:Novartis: Consultancy, Research Funding; Astellas Pharma: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Arog: Research Funding. Ossenkoppele:Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Research Funding; Roche: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; Johnson & Johnson: Consultancy, Honoraria, Research Funding; Genmab: Research Funding.

A phase I/II study combining tosedostat with capecitabine in patients with metastatic pancreatic ductal adenocarcinoma (PDAC).

410 Background: Recent advances in front-line therapy have improved survival in patients with advanced PDAC. A fluorouracil-based regimen is recommended for patients who progress on a gemcitabine-based therapy. Tosedostat is an oral aminopeptidase inhibitor that disrupts the cleavage of amino acids from peptides downstream of proteasomal degradation. It prevents the recycling of free amino acids, leads to intracellular depletion of amino acids, and triggers an amino acid deprivation response that subsequently results in apoptosis. Because PDAC cells frequently upregulate expression of these aminopeptidases, tosedostat offers therapeutic promise, particularly in combination with fluoropyrimidines. Methods: This is a single institution phase I/II trial to evaluate the safety and toxicity of tosedostat plus capecitabine in patients with metastatic PDAC progressed on a gemcitabine-based therapy. The phase I portion is being conducted in a dose de-escalation fashion, with two dose levels of tosedostat (120 mg or 60 mg) p.o. daily on days 1 to 21 with capecitabine 1000 mg/m2 p.o. BID on days 1 to 14 of a 21-day cycle. If more than one out of 6 patients in the tosedostat (120 mg) cohort experience a dose limiting toxicity (DLT), then 6 more will be enrolled to the tosedostat (60 mg) cohort. The primary objective of the phase I portion is to determine the optimal phase II dose. The primary objective of the phase II portion is to determine the progression-free survival at 3 months. Secondary objectives include the overall response rate, overall survival and CA 19-9 response. To avoid futility, interim analysis is planned after 10 evaluable patients enrolled. Results: Up to date, a total of 11 patients have been enrolled in the study, and 10 patients are evaluable. No DLT have been observed. Tosedostat at a dose of 120 mg with capecitabine is extremely well tolerated. Prolonged stable disease has been observed in 4 (40%) patients with a time on treatment of 10 months, 7.5 months, 5.5 months and 4 months, and 3 of the 4 patients remain on the trial. Conclusions: The combination of tosedostat and capecitabine is a well-tolerated regimen with impressive clinical activity in the subset of patients studied thus far. Clinical trial information: NCT02352831.

A phase I/II study combining tosedostat with capecitabine in patients with metastatic pancreatic adenocarcinoma.

TPS471 Background: Metastatic pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. Recent advances with fluorouracil in combination with oxaliplatin and irinotecan (FOLFIRINOX) and nab-paclitaxel combined with gemcitabine (AG) have improved survival in patients with PDAC. A fluorouracil-based regimen is recommended for patients who progress after a gemcitabine-based regimen. Tosedostat is an oral aminopeptidase inhibitor shown to have anti-proliferative effects in malignancies. Aminopeptidase inhibitors disrupt the cleavage of amino acids from peptides downstream of proteasomal degradation, preventing the recycling of amino acids needed for new protein synthesis. This leads to intracellular depletion of amino acids, resulting in a cellular stress response known as the amino acid deprivation response, which leads to apoptosis. Because pancreatic cancer cells frequently upregulate expression of these aminopeptidases, aminopeptidases inhibitors hold therapeutic promise. Methods: This is a single institution phase I/II open-label trial to evaluate the safety and tolerability of tosedostat plus capecitabine in patients with metastatic PDAC that have progressed after a gemcitabine-based regimen. The phase I part will be conducted in a dose de-escalation fashion, with two planned dose levels of tosedostat (120mg or 60mg) p.o. daily on days 1 to 21 with capecitabine 1000 mg/m2 p.o. BID on days 1 to 14 of a 21-day cycle. If more than one patient in the tosedostat (120 mg) cohort experiences a dose limiting toxicity (DLT), then 6 more patient will be enrolled to the tosedostat (60 mg) cohort. A total of 36 patients will be enrolled in the phase II portion. Primary objective of the phase I portion is to determine the maximum tolerated dose and DLTs of tosedostat and capecitabine combination therapy. Primary objective of the phase II portion is to determine the progression-free survival at 3 months. Secondary objectives are to determine the overall response rate, time-to-progression, overall survival and CA 19-9 response. Exploratory objectives are to explore the predictive molecular biomarkers for treatment response and to explore the prognostic biomarkers. Clinical trial: NCT02352831. Clinical trial information: NCT02352831.

Activation of the antioxidant response in methionine deprived human cells results in an HSF1-independent increase in HSPA1A mRNA levels

In cells starved for leucine, lysine or glutamine heat shock factor 1 (HSF1) is inactivated and the level of the transcripts of the HSF1 target genes HSPA1A (Hsp70) and DNAJB1 (Hsp40) drops. We show here that in HEK293 cells deprived of methionine HSF1 was similarly inactivated but that the level of HSPA1A and DNAJB1 mRNA increased. This increase was also seen in cells expressing a dominant negative HSF1 mutant (HSF379 or HSF1-K80Q), confirming that the increase is HSF1 independent. The antioxidant N-acetylcysteine completely inhibited the increase in HSPA1A and DNAJB1 mRNA levels upon methionine starvation, indicating that this increase is a response to oxidative stress resulting from a lack of methionine. Cells starved for methionine contained higher levels of c-Fos and FosB mRNA, but knockdown of these transcription factors had no effect on the HSPA1A or DNAJB1 mRNA level. Knockdown of NRF2 mRNA resulted in the inhibition of the increase in the HSPA1A mRNA, but not the DNAJB1 mRNA, level in methionine starved cells. We conclude that methionine deprivation results in both the amino acid deprivation response and an antioxidant response mediated at least in part by NRF2. This antioxidant response includes an HSF1 independent increase in the levels of HSPA1A and DNAJB1 mRNA.

Targeting Aminopeptidases by Tosedostat (TST) (CHR2797), Alone and with LBH589, Induces Significant Cytotoxicity Against Human Multiple Myeloma (MM) Cells

AbstractAbstract 1847Aminopeptidases (AP) are necessary for the growth and development of malignant cells and have a selectively important role in the maintenance of intracellular amino acid (AA) levels in neoplastic cells. CHR2797 is a novel, low nanomolar inhibitor of the M1 family of AP, a group of metalloenzymes containing a central Zn2+ ion. CHR2797 has antiproliferative and apoptotic effects against MM in vitro by inducing the AA deprivation response (AADR). TST, an oral, chronically administered agent with a good safety profile has demonstrated activity in patients with relapsed/refractory AML and is currently under study as part of combination therapy for untreated elderly patients with AML. At the epigenetic regulatory level, Zn-dependent histone deacetylase (HDAC) cause the deacetylation of histone and non-histone cellular proteins which are critical for gene expression, inducing apoptosis and cell cycle arrest in cancer cells. LBH589 (Panobinostat) is an established pan-HDAC inhibitor with potent in vitro anti-cancer activity in many hematological malignancies. The clinical efficacy of Panobinostat is currently being studied in several Phase II/III clinical trials with particular promise seen in the treatment of MM. Here we examined the potential therapeutic effect of CHR2797, alone and with LBH589, against MM cells. Using MTS and CTG assays, CHR2797, at clinically achievable concentrations, decreased survival and proliferation in MM1S and IL-6-dependent ANBL6 cells, in the presence or absence of bone marrow stromal cells following 72 hours incubation. CHR2797 induces apoptosis in MM cells via activation of Caspase 3/7 and 9 but not Caspase 8. Significantly, CHR2797 (10 μM) induced apoptosis in patient MM cells, as seen by % of annexin V and PI from 22 + 1.5% to 39 + 2.3% after 48h incubation. Combined treatment with CHR2797 and LBH589 in MM cells (MM1S, ANBL6, and INA6) further reduced cell viability following 72 hour incubation when compared with CHR2797 treatment alone, as determined by CTG viability luminescent assay. Both drugs together also augmented growth inhibitory effects when compared with single agent alone, after 72 hours incubation followed by MTS assay. Importantly, the combination of both drugs increased caspase 3/7- & 9-mediated apoptosis than CHR2797 alone in these MM cells following 24h-treatment. Cell cycle analysis (CHR2797 at 1μM; LBH589 at 1 nM) showed an increased growth arrest in G0/G1 cells in MM1R cells treated with both drugs versus CHR2797 alone after 24 hours: 68.5±3.3% versus 36±2.5%. Furthermore, CHR2797 inhibited anti-apoptotic protein Mcl-1 in MM1R and U266 MM cells by immunoblottings. Combined treatment with CHR2797 and LBH589 further blocked Mcl-1 when compared with either treatment alone after 24 hours incubation. Together, these results show that the combination of CHR2797 and LBH589 enhanced anti-myeloma effects when compared with either drug alone. This combination, which also has the potential of being without overlapping clinical toxicities, provides a promising novel approach to anti-myeloma therapy. Disclosures:Singer:Cell Therapeutics, Inc: Employment, Equity Ownership. Richardson:Novartis: Membership on an entity's Board of Directors or advisory committees.

Quantitative proteomics and metabolomics approaches to demonstrate N-acetyl-d-glucosamine inducible amino acid deprivation response as morphological switch in Candida albicans

Candida albicans is a life threatening polymorphic pathogen for immunocompromised patients, causing superficial as well as invasive systemic diseases. The mucosal membranes of the host, which are the primary sites of its infection, are rich in amino sugars like N-acetylglucosamine (GlcNAc). GlcNAc is also one of the potent inducers of morphological transition, an important pathogenic trait of C. albicans. We thus performed proteomic analysis on total soluble proteins to identify the molecules involved in this response. Proteomic analysis using 2-DE demonstrated reproducible upregulation of 36 spots from a total of 585 matched spots. Mass spectroscopy (MS/MS) analyses of upregulated proteins revealed that carbohydrate and amino acid metabolism were the most prominent functional classes. Metabolite profiling using GC–MS allowed a quantitative comparison of 58 metabolites in GlcNAc or glucose grown cells. We observed a significant decrease in the intracellular amino acid pool of GlcNAc grown cells. Moreover, GlcNAc induces both bZIP transcription factor (GCN4) and eIF2α kinase (GCN2) which are responsible for the activation of general amino acid control response in C. albicans. Inactivation of these genes blocks GlcNAc induced morphogenesis. Altogether these results suggest that amino acid starvation is the morphogenetic signal in presence of GlcNAc in C. albicans.

Results of the OPAL Study: A Phase II Study to Evaluate the Efficacy, Safety and Tolerability of Tosedostat (CHR-2797) in Elderly Subjects with Treatment Refractory or Relapsed Acute Myeloid Leukemia

Abstract 767 Background:Tosedostat is a novel oral inhibitor of the M1/17 family of aminopeptidases which induces an amino acid deprivation response that is selectively toxic for myeloid blasts (Leuk Res. 2011: 5:677-81) and has shown promising activity in elderly relapsed/refractory AML patients (J Clin Oncol 2010:28:4333-8). Aims:The OPAL study was undertaken to compare the activity of tosedostat at a once-daily dose of 120 mg for 24 weeks compared to 240 mg once daily for 8 weeks followed by 120 mg once daily for a further 16 weeks., as measured by bone marrow and hematology responses at 24 weeks. Methods:This was a phase II randomized (1:1) multi-center study. Patients were eligible if aged 60 years or older with previous CR lasting <12 months, or no CR after primary therapy, had a peripheral blast count <30,000/μl, PS<2 and adequate renal, hepatic and cardiac function. The primary analysis was performed at 24 weeks using IWG 2003 criteria. Results:Seventy-three patients were randomized and received tosedostat, 38 at 120 mg and 35 at 240 mg. Median age was 72 (range, 64 to 86), and 59% were male. Twenty-six patients (36%) had secondary or therapy-related AML, of which 19 (26%) had prior MDS. Median time since AML diagnosis was 211 days and 38% had received primary therapy with cytarabine/anthracyclines; 36% with a hypomethylating agent (HMA) and 23% with other cytarabine regimes. Fifty-two percent had been refractory to primary therapy, 19% had previously had a remission of up to 6 months and 29% a 6–12 month remission (mean 97 days including refractory). Twenty-three patients (32%) had no post-treatment bone marrow sample taken, predominantly due to early progression: 34% completed 12 weeks on study and 14% completed 24 weeks and were eligible to enter an extension study which is ongoing. The overall response rate was 22%; (CR/CRp/MLFS 12%; PR 10%) and an additional 29% had a best response of stable disease. The most common adverse events which occurred (total; grade 3 or worse) were diarrhea (58%; 4.1%), peripheral edema (55%; 0%), fatigue (49%; 21%), dyspnea (41%; 16%), nausea (38%; 0%), decreased appetite (37%; 3%), febrile neutropenia (36%; 29%) and hypotension (36%, 10%). Median overall survival (OS) (at 15 July 2011) was 126 days. Median OS in patients with CR/CRp/MLFS, PR and SD were 280, 195 and 162 days respectively, and 261.5 days for patients with a response of PR or better. Median OS for patients with progression of disease or who were unevaluable was 61 days. Similar responses were seen in the two dose groups. Additional non protocol-specified analyses showed that the following types of patient appeared to respond well: AML NOC vs other AML types 16% vs 29% response, median OS 75 vs 168 days; patients with poor risk cytogenetics compared to intermediate/better, median OS 159 vs 107 days; those who received prior HMA compared to others, 38% vs 13% response, median OS 171 vs 104 days; and absence of prior CR 29% vs 14% response and median OS 169 vs 103 days. Conclusions:These results provide further encouraging evidence of efficacy and a favorable toxicity profile in a difficult to treat patient population. A phase III program of pivotal studies with tosedostat in AML and MDS will start in the near future.Response Category (n=73)Number (%)Overall Survival (median days)All Patientsn/a126 (4–452)Overall Responder (PR or better)16 (22%)261.5 (71–445)CR/CRp/MLFS9 (12%)280 (181–422)Partial Remission7 (10%)195 (71–445)Stable Disease21 (29%)162 (43–284)Progressive Disease/unevaluable36 (49%)61 (4–452) Disclosures:Cortes:Chroma Therapeutics Ltd.: Consultancy, Research Funding. Feldman:Chroma Therapeutics Ltd.: Consultancy, Research Funding. Yee:Chroma Therapeutics Ltd.: Consultancy, Research Funding. Rizzieri:Chroma Therapeutics Ltd.: Consultancy, Research Funding. Advani:Chroma Therapeutics Ltd.: Consultancy, Research Funding. Charman:Chroma Therapeutics Ltd.: Employment, Equity Ownership. Toal:Chroma Therapeutics Ltd.: Employment, Equity Ownership. Kantarjian:Chroma Therapeutics Ltd.: Consultancy, Research Funding.

509 The OPAL Study: A Phase II Study to Evaluate the Efficacy, Safety and Tolerability of Tosedostat (CHR-2797) in Elderly Subjects with Treatment Refractory or Relapsed Acute Myeloid Leukemia

Abstract Abstract 767 Background: Tosedostat is a novel oral inhibitor of the M1/17 family of aminopeptidases which induces an amino acid deprivation response that is selectively toxic for myeloid blasts (Leuk Res. 2011: 5:677-81) and has shown promising activity in elderly relapsed/refractory AML patients (J Clin Oncol 2010:28:4333-8). Aims: The OPAL study was undertaken to compare the activity of tosedostat at a once-daily dose of 120 mg for 24 weeks compared to 240 mg once daily for 8 weeks followed by 120 mg once daily for a further 16 weeks., as measured by bone marrow and hematology responses at 24 weeks. Methods: This was a phase II randomized (1:1) multi-center study. Patients were eligible if aged 60 years or older with previous CR lasting Results: Seventy-three patients were randomized and received tosedostat, 38 at 120 mg and 35 at 240 mg. Median age was 72 (range, 64 to 86), and 59% were male. Twenty-six patients (36%) had secondary or therapy-related AML, of which 19 (26%) had prior MDS. Median time since AML diagnosis was 211 days and 38% had received primary therapy with cytarabine/anthracyclines; 36% with a hypomethylating agent (HMA) and 23% with other cytarabine regimes. Fifty-two percent had been refractory to primary therapy, 19% had previously had a remission of up to 6 months and 29% a 6–12 month remission (mean 97 days including refractory). Twenty-three patients (32%) had no post-treatment bone marrow sample taken, predominantly due to early progression: 34% completed 12 weeks on study and 14% completed 24 weeks and were eligible to enter an extension study which is ongoing. The overall response rate was 22%; (CR/CRp/MLFS 12%; PR 10%) and an additional 29% had a best response of stable disease. The most common adverse events which occurred (total; grade 3 or worse) were diarrhea (58%; 4.1%), peripheral edema (55%; 0%), fatigue (49%; 21%), dyspnea (41%; 16%), nausea (38%; 0%), decreased appetite (37%; 3%), febrile neutropenia (36%; 29%) and hypotension (36%, 10%). Median overall survival (OS) (at 15 July 2011) was 126 days. Median OS in patients with CR/CRp/MLFS, PR and SD were 280, 195 and 162 days respectively, and 261.5 days for patients with a response of PR or better. Median OS for patients with progression of disease or who were unevaluable was 61 days. Similar responses were seen in the two dose groups. Additional non protocol-specified analyses showed that the following types of patient appeared to respond well: AML NOC vs other AML types 16% vs 29% response, median OS 75 vs 168 days; patients with poor risk cytogenetics compared to intermediate/better, median OS 159 vs 107 days; those who received prior HMA compared to others, 38% vs 13% response, median OS 171 vs 104 days; and absence of prior CR 29% vs 14% response and median OS 169 vs 103 days. Conclusions: These results provide further encouraging evidence of efficacy and a favorable toxicity profile in a difficult to treat patient population. A phase III program of pivotal studies with tosedostat in AML and MDS will start in the near future. Response Category (n=73) Number (%) Overall Survival (median days) All Patients n/a 126 (4–452) Overall Responder (PR or better) 16 (22%) 261.5 (71–445) CR/CRp/MLFS 9 (12%) 280 (181–422) Partial Remission 7 (10%) 195 (71–445) Stable Disease 21 (29%) 162 (43–284) Progressive Disease/unevaluable 36 (49%) 61 (4–452) Disclosures: Cortes: Chroma Therapeutics Ltd.: Consultancy, Research Funding. Feldman: Chroma Therapeutics Ltd.: Consultancy, Research Funding. Yee: Chroma Therapeutics Ltd.: Consultancy, Research Funding. Rizzieri: Chroma Therapeutics Ltd.: Consultancy, Research Funding. Advani: Chroma Therapeutics Ltd.: Consultancy, Research Funding. Charman: Chroma Therapeutics Ltd.: Employment, Equity Ownership. Toal: Chroma Therapeutics Ltd.: Employment, Equity Ownership. Kantarjian: Chroma Therapeutics Ltd.: Consultancy, Research Funding.

Expression profiling after activation of amino acid deprivation response in HepG2 human hepatoma cells

Dietary protein malnutrition is manifested as amino acid deprivation of individual cells, which activates an amino acid response (AAR) that alters cellular functions, in part, by regulating transcriptional and posttranscriptional mechanisms. The AAR was activated in HepG2 human hepatoma cells, and the changes in mRNA content were analyzed by microarray expression profiling. The results documented that 1,507 genes were differentially regulated by P < 0.001 and by more than twofold in response to the AAR, 250 downregulated and 1,257 upregulated. The spectrum of altered genes reveals that amino acid deprivation has far-reaching implications for gene expression and cellular function. Among those cellular functions with the largest numbers of altered genes were cell growth and proliferation, cell cycle, gene expression, cell death, and development. Potential biological relationships between the differentially expressed genes were analyzed by computer software that generates gene networks. Proteins that were central to the most significant of these networks included c-myc, polycomb group proteins, transforming growth factor β1, nuclear factor (erythroid-derived 2)-like 2-related factor 2, FOS/JUN family members, and many members of the basic leucine zipper superfamily of transcription factors. Although most of these networks contained some genes that were known to be amino acid responsive, many new relationships were identified that underscored the broad impact that amino acid stress has on cellular function.

Inhibition of HDACs and Aminopeptidases Is Highly Synergistic in Myeloma Cells Resulting in Cell Death Via the Upregulation of BIRC3, a Key Mediator of NF-KappaB Signalling.

Abstract 607Myeloma cells rely on a number of mechanisms to maintain their survival, including the silencing of genes that would normally check uncontrolled proliferation and lead to apoptosis. Epigenetic alterations such as histone deacetylation contribute significantly to the pathogenesis of both solid and haematological malignancies and are associated with the silencing of tumour suppressor genes. Pan-histone deacetylase inhibitors (HDACs), such as SAHA, panobinostat, depsipeptide, and numerous others have been published to have anti-myeloma activity. Here we report the effects of a novel pan HDAC inhibitor, CHR-3996, on multiple myeloma cells. Results: CHR-3996 was potently cytotoxic against a panel of myeloma cell lines (H929, KMS11, LP-1, MM1s, RPMI-8226, and U266) with a GI50 ranging from 9 to 65 nM and was equally as potent in primary patient myeloma cells. The loss of cell viability was associated with an increase in apoptotic cells; EndonucleaseG and Noxa were both up-regulated and caspase 9 was cleaved. Furthermore a decrease in apoptosis was demonstrated in the presence of a pan-caspase inhibitor indicating cell death is largely dependent on caspase-mediated apoptosis. There was no effect on bone marrow stromal (BMS) cell viability but there was an observed decrease in the IL-6 and VEGF the BMS cells secrete, both of which promote the growth and survival of myeloma cells in the bone marrow microenvironment. CHR-3996 caused an increase in acetylated H3K9 but there was minimal change in the levels of ubiquitinated proteins in the cell or acetylated alpha-tubulin, indicating low activity against HDAC6 or the proteasome (also demonstrated by an assay to specifically measure proteasome function). In addition to induction of apoptosis, cell cycle analysis showed an increased proportion of cells in G0/G1 indicating cell cycle arrest. In-keeping with data from treating myeloma cells with SAHA and panobinostat, an increase in cell cycle inhibitor p21 was observed. Gene expression profiling (GEP) identified changes in cell cycle regulators, indications of increased cell stress (elevated CHOP, ATF3, and TAO kinase 3), repression of Wnt (up-regulation of NLK, GSK3beta) and mTOR (decreased 4E-BP1) signalling, and changes in key pro- and anti-apoptotic proteins (for example SMAD3, BCL2, BIM, BID, and BIRC5). When used in combination studies CHR-3996 was highly synergistic in vitro with tosedostat, an aminopeptidase inhibitor, which we have previously shown to have anti-myeloma activity via the induction of the amino acid deprivation response and autophagy. One of the largest changes the GEP analysis identified was BIRC3 (CIAP2), an inhibitor of NF-kappaB signalling, which increased 23.5 fold with CHR-3996 as a single agent and over 100-fold when added to H929 cells in combination with tosedostat. Additionally both CHR-3996 and todestat independently up-regulated expression of members of the IkappaB family. Altered expression and nuclear localisation of canonical and non-canonical NF-kappaB family members were observed by immuno-fluorescence and immunoblotting, suggesting targeting of NF-kappaB signalling as a reason for the high degree of synergy between these two compounds. Early data suggest oral CHR-3996 is effective in a subcutaneous in vivo myeloma model and, reflecting the in vitro data, there is a degree of synergy when administered with tosedostat. Conclusions: The novel compound CHR-3996 is a potent HDAC inhibitor that leads to increased H3K9 acetylation but has no detectable activity against HDAC6 or proteasome activity in myeloma. It induces apoptosis of myeloma cells without affecting BMS cell viability. CHR-3996 shows a very high degree of synergy with an aminopeptidase inhibitor, tosedostat, potentially through targeting the NF-kappaB pathway. It has exciting therapeutic potential either as a mono-therapy or in combination with other agents. Disclosures:Krige:Chroma Therapeutics Ltd: Employment, Equity Ownership. Hooftman:Chroma Therapeutic Ltd: Employment, Equity Ownership. Drummond:Chroma therapuetics: Employment, Equity Ownership.

Aminopeptidase inhibition as a targeted treatment strategy in myeloma

Myeloma cells are highly dependent on the unfolded protein response to assemble folded immunoglobulins correctly. Therefore, targeting protein handling within a myeloma cell by inhibiting the aminopeptidase enzyme system, which catalyses the hydrolysis of amino acids from the proteins NH2 terminus, represents a therapeutic approach. CHR-2797, a novel aminopeptidase inhibitor, is able to inhibit proliferation and induce growth arrest and apoptosis in myeloma cells, including cells resistant to conventional chemotherapeutics. It causes minimal inhibition of bone marrow stromal cell (BMSC) proliferation but is able to overcome the microenvironmental protective effects, inhibiting the proliferation of myeloma cells bound to BMSCs and the increase in vascular endothelial growth factor levels seen when myeloma cells and BMSCs are bound together. Additive and synergistic effects are seen with bortezomib, melphalan, and dexamethasone. Apoptosis occurs via both caspase-dependent and non-caspase-dependent pathways with an increase in Noxa, cleavage of Mcl-1, and activation of the unfolded protein response. Autophagy is also seen. CHR-2797 causes an up-regulation of genes involved in the proteasome/ubiquitin pathway, as well as aminopeptidases, and amino acid deprivation response genes. In conclusion, inhibiting protein turnover using the aminopeptidase inhibitor CHR-2797 results in myeloma cell apoptosis and represents a novel therapeutic approach that warrants further investigation in the clinical setting.

CHR-2797: An Antiproliferative Aminopeptidase Inhibitor that Leads to Amino Acid Deprivation in Human Leukemic Cells

CHR-2797 is a novel metalloenzyme inhibitor that is converted into a pharmacologically active acid product (CHR-79888) inside cells. CHR-79888 is a potent inhibitor of a number of intracellular aminopeptidases, including leucine aminopeptidase. CHR-2797 exerts antiproliferative effects against a range of tumor cell lines in vitro and in vivo and shows selectivity for transformed over nontransformed cells. Its antiproliferative effects are at least 300 times more potent than the prototypical aminopeptidase inhibitor, bestatin. However, the mechanism by which inhibition of these enzymes leads to proliferative changes is not understood. Gene expression microarrays were used to profile changes in mRNA expression levels in the human promyelocytic leukemia cell line HL-60 treated with CHR-2797. This analysis showed that CHR-2797 treatment induced a transcriptional response indicative of amino acid depletion, the amino acid deprivation response, which involves up-regulation of amino acid synthetic genes, transporters, and tRNA synthetases. These changes were confirmed in other leukemic cell lines sensitive to the antiproliferative effects of CHR-2797. Furthermore, CHR-2797 treatment inhibited phosphorylation of mTOR substrates and reduced protein synthesis in HL-60 cells, both also indicative of amino acid depletion. Treatment with CHR-2797 led to an increase in the concentration of intracellular small peptides, the substrates of aminopeptidases. It is suggested that aminopeptidase inhibitors, such as CHR-2797 and bestatin, deplete sensitive tumor cells of amino acids by blocking protein recycling, and this generates an antiproliferative effect. CHR-2797 is orally bioavailable and currently undergoing phase II clinical investigation in the treatment of myeloid leukemia.

Cellular responses to cysteine deprivation are mediated by the amino acid deprivation pathway but not the oxidative stress pathway.

Several genes in cysteine (Cys) metabolism are transcriptionally regulated in response to either Cys deprivation or oxidative stress. These include GCLC and GCLM, which encode glutamate:Cys ligase subunits that catalyze the first step of glutathione synthesis, and SLC7A11, which encodes the cystine‐glutamate exchanger responsible for cystine uptake. In Cys‐deprived HepG2/C3A cells, GCLC, GCLM and SLC7A11 were upregulated as assessed by microarray analysis and qRT‐PCR. Expression of many genes with antioxidant response elements was not affected by Cys deprivation while genes with amino acid response elements (AAREs) were consistently upregulated, suggesting that Cys deprivation can induce the eIF2α kinase 4 mediated amino acid deprivation pathway without inducing oxidative stress. Induction of GCLC, GCLM and SLC7A11 was also observed in rats fed 10% (w/w) protein diets, and activation of the AARE‐mediated pathway was evidenced by phosphorylation of eIF2α and upregulation of mRNA and protein levels for genes with AAREs. The pattern of gene expression in growing rats fed diets marginally deficient in sulfur amino acids suggests that the amino acid deprivation response can be activated in response to mild, as well as severe, amino acid deficiencies. SLC7A11 contains an AARE and is likely a direct target, but the details of the regulation of GCLC and GCLM expression in response to Cys deficiency need further study. Funded by NIH Grant PHS DK0664303.

Aminopeptidase Inhibition as a Targeted Treatment Strategy in Myeloma.

Myeloma cells are highly dependent on the unfolded protein response to assemble folded immunoglobulins correctly. Therefore targeting protein handling within a myeloma cell by inhibiting the aminopeptidase enzyme system that catalyses the hydrolysis of amino acids from the N terminus of proteins may be a novel therapeutic approach. The effect of the aminopeptidase inhibitor CHR-2797 on myeloma cell proliferation and survival, gene expression, protein turnover, cell migration and myeloma-bone marrow stromal cell interactions was determined on a panel of myeloma cell lines and patient cells. CHR-2797 is able to inhibit the proliferation of myeloma cell lines and primary patient cells, whereas its derivative CHR-79888, an acid metabolite with low cell membrane permeability fails to induce myeloma cell death. This occurs though apoptosis as demonstrated by trypan blue exclusion and annexin V/PI staining, and is proceeded by G1 growth arrest. Western blot analysis demonstrates apoptosis occurs via a non-caspase dependant mechanism. Importantly CHR-2797 is able to induce apoptosis in cells known to be resistant to conventional chemotherapeutic agents. Analysis of the pathways involved using Affymetrix gene expression arrays demonstrates CHR-2797 causes an upregulation of many genes involved in the proteasome/ubiquitin pathway, as well as amino acid deprivation response genes and some aminopeptidases. A further mechanism contributing to cell death is activation of the unfolded protein response with activation of all three UPR pathways demonstrated by splicing of XBP1 to its active from XBP1s, an increase in CHOP with activation of the PERK pathway and cleavage of ATF6. Cytoplasmic inclusions are also present on light microscopy suggestive of the build up of misfolded proteins within the cytoplasm. CHR-2797 causes minimal inhibition of the proliferation of bone marrow stroma, but is able to overcome the protective effects of the micro-environment on myeloma cells, as the drug is still able to inhibit the proliferation of myeloma cells when they are bound to bone marrow stromal cells. Aminopeptidase inhibition is also able to inhibit the increase VEGF that occurs when myeloma cells and bone marrow stroma are bound together. Combination experiments of CHR-2797 with dexamethasone demonstrate synergy, in keeping with the different mechanisms of action the two drugs. CHR-2797 in combination with the proteasome inhibitor bortezomib demonstrates an additive effect. Although both drugs target intracellular protein turnover, gene expression studies of cells treated with CHR-2797 or bortezomib show deregulation of a number of genes specific to aminopeptidase inhibition, as well as a series of genes characteristic of protein turnover. These differences in the mechanism of action of the two drugs are also reflected in the Western blot analysis that demonstrates a predominately non-caspase mediated cell death in CHR2797 compared to a caspase mediated cell death with bortezomib. In summary inhibiting intracellular protein turnover using the aminopeptidase inhibitor CHR-2797 results in myeloma cell death and represents a novel therapeutic approach for the treatment of myeloma. A phase 1 clinical trial has been initiated in haematological malignancies and the results will also be presented at this meeting.

Integrated Proteomics and Genomics Strategies Bring New Insight into Candida albicans Response upon Macrophage Interaction

The interaction of Candida albicans with macrophages is considered a crucial step in the development of an adequate immune response in systemic candidiasis. An in vitro model of phagocytosis that includes a differential staining procedure to discriminate between internalized and non-internalized yeast was developed. Upon optimization of a protocol to obtain an enriched population of ingested yeasts, a thorough genomics and proteomics analysis was carried out on these cells. Both proteins and mRNA were obtained from the same sample and analyzed in parallel. The combination of two-dimensional PAGE with MS revealed a total of 132 differentially expressed yeast protein species upon macrophage interaction. Among these species, 67 unique proteins were identified. This is the first time that a proteomics approach has been used to study C. albicans-macrophage interaction. We provide evidence of a rapid protein response of the fungus to adapt to the new environment inside the phagosome by changing the expression of proteins belonging to different pathways. The clear down-regulation of the carbon-compound metabolism, plus the up-regulation of lipid, fatty acid, glyoxylate, and tricarboxylic acid cycles, indicates that yeast shifts to a starvation mode. There is an important activation of the degradation and detoxification protein machinery. The complementary genomics approach led to the detection of specific pathways related to the virulence of Candida. Network analyses allowed us to generate a hypothetical model of Candida cell death after macrophage interaction, highlighting the interconnection between actin cytoskeleton, mitochondria, and autophagy in the regulation of apoptosis. In conclusion, the combination of genomics, proteomics, and network analyses is a powerful strategy to better understand the complex host-pathogen interactions.