Median IC50 Research Articles

Selective BCL-2 Inhibition by ABT-199 Causes On-Target Cell Death in Acute Myeloid Leukemia

B-cell leukemia/lymphoma 2 (BCL-2) prevents commitment to programmed cell death at the mitochondrion. It remains a challenge to identify those tumors that are best treated by inhibition of BCL-2. Here, we demonstrate that acute myeloid leukemia (AML) cell lines, primary patient samples, and murine primary xenografts are very sensitive to treatment with the selective BCL-2 antagonist ABT-199. In primary patient cells, the median IC50 was approximately 10 nmol/L, and cell death occurred within 2 hours. Our ex vivo sensitivity results compare favorably with those observed for chronic lymphocytic leukemia, a disease for which ABT-199 has demonstrated consistent activity in clinical trials. Moreover, mitochondrial studies using BH3 profiling demonstrate activity at the mitochondrion that correlates well with cytotoxicity, supporting an on-target mitochondrial mechanism of action. Our protein and BH3 profiling studies provide promising tools that can be tested as predictive biomarkers in any clinical trial of ABT-199 in AML.

Initial testing (stage 1) of the investigational mTOR kinase inhibitor MLN0128 by the pediatric preclinical testing program

MLN0128 is an investigational small molecule ATP-competitive inhibitor of the serine/threonine kinase mTOR. MLN0128 was tested against the in vitro panel at concentrations ranging from 0.1 nM to 1 μM and against the PPTP in vivo panels at a dose of 1 mg/kg administered orally daily × 28. In vitro the median relative IC(50) concentration was 19 nM. In vivo MLN0128 induced significant differences in EFS in 24/31 (77%) solid tumor models, but 0/7 ALL xenografts. The modest activity observed for MLN0128 against the PPTP preclinical models is similar to that previously reported for another TOR kinase inhibitor.

Four years’ monitoring of in vitro sensitivity and candidate molecular markers of resistance of Plasmodium falciparum to artesunate-mefloquine combination in the Thai-Myanmar border

BackgroundThe decline in efficacy of artesunate (AS) and mefloquine (MQ) is now the major concern in areas along the Thai-Cambodian and Thai-Myanmar borders.MethodsThe correlation between polymorphisms of pfatp6, pfcrt, pfmdr1 and pfmrp1 genes and in vitro sensitivity of Plasmodium falciparum isolates to the artemisinin-based combination therapy (ACT) components AS and MQ, including the previously used first-line anti-malarial drugs chloroquine (CQ) and quinine (QN) were investigated in a total of 119 P. falciparum isolates collected from patients with uncomplicated P. falciparum infection during 2006–2009.ResultsReduced in vitro parasite sensitivity to AS [median (95% CI) IC50 3.4 (3.1-3.7) nM] was found in 42% of the isolates, whereas resistance to MQ [median (95% CI) IC50 54.1 (46.8-61.4) nM] accounted for 58% of the isolates. Amplification of pfmdr1 gene was strongly associated with a decline in susceptibility of P. falciparum isolates to AS, MQ and QN. Significant difference in IC50 values of AS, MQ and QN was observed among isolates carrying one, two, three, and ≥ four gene copies [median (95% CI) AS IC50: 1.6 (1.3-1.9), 1.8 (1.1-2.5), 2.9 (2.1-3.7) and 3.1 (2.5-3.7) nM, respectively; MQ IC50: 19.2 (15.8-22.6), 37.8 (10.7-64.8), 55.3 (47.7-62.9) and 63.6 (49.2-78.0) nM, respectively; and QN IC50: 183.0 (139.9-226.4), 256.4 (83.7-249.1), 329.5 (206.6-425.5) and 420.0 (475.2-475.6) nM, respectively]. The prevalence of isolates which were resistant to QN was reduced from 21.4% during the period 2006–2007 to 6.3% during the period 2008–2009. Pfmdr1 86Y was found to be associated with increased susceptibility of the parasite to MQ and QN. Pfmdr1 1034C was associated with decreased susceptibility to QN. Pfmrp1 191Y and 1390I were associated with increased susceptibility to CQ and QN, respectively.ConclusionHigh prevalence of CQ and MQ-resistant P. falciparum isolates was observed during the four-year observation period (2006–2009). AS sensitivity was declined, while QN sensitivity was improved. Pfmdr1 and pfmrp1 appear to be the key genes that modulate multidrug resistance in P. falciparum.

The in vitro antimalarial interaction of 9-hydroxycalabaxanthone and α-mangostin with mefloquine/artesunate.

Multidrug resistance Plasmodium falciparum is the major health problem in Thailand. Discovery and development of new antimalarial drugs with novel modes of action is urgently required. The aim of the present study was to investigate the antimalarial interaction of 9-hydroxycalabaxanthone and α-mangostin with the standard antimalarial drugs mefloquine and artesunate in chloroquine sensitive (3D7) and chloroquine resistant (K1) P. falciparum clones in vitro. Median (range) IC50 (drug concentration which produces 50% parasite growth inhibition) values of the 9-hydroxycalabaxanthone, α-mangostin, artesunate and mefloquine for 3D7 vs K1 clones were 1.5 (0.9-2.1) vs 1.2 (1.1-1.6) μM, 17.9 (15.7.0-20.0) vs 9.7 (6.0-14.0) μM, 1.0 (0.4-3.0) vs 1.7 (1.0-2.5) nM, and 13.3 (11.1-13.3) vs 7.1 (6.7-12.2) nM, respectively. Analysis of isobologram and combination index (CI) of 9-hydroxycalabaxanthone with artesunate or mefloquine showed synergistic and indifference antimalarial interaction, respectively. α-mangostin-artesunate combination exhibited a slight antagonistic effect of antimalarial interaction, whereas α-mangostin and mefloquine combination showed indifference interaction in both clones. The combination of 9-hydroxycalabaxanthone with α-mangostin showed the synergistic antimalarial interaction in both clones.

In-vitro phenotypic susceptibility of HIV-1 ’non-B’ integrase inhibitors naive clinical isolates to dolutegravir and raltegravir

In this study, we assessed phenotypic susceptibility to dolutegravir and raltegravir in a large variety of HIV-1 'non-B' subtypes (n = 72) issued from integrase inhibitor-naive clinical isolates. All samples were susceptible to both dolutegravir and raltegravir with median IC50 values of 1.22 nmol/l and 1.53 nmol/l, respectively; similar to that observed for the B subtype. Thus, despite the high prevalence of polymorphic substitutions in integrase in 'non-B' clinical isolates, phenotypic susceptibility to dolutegravir remained unchanged.

PQJS380

Acute lymphoblastic leukemia (ALL) is a malignant disorder of lymphoid progenitor cells that are committed to the B- or the T-cell lineage. The pathogenesis of ALL is heterogeneous and may be at least in part caused by genetic alterations. Although the modern sequencing technologies make it possible to rapidly discover novel genetic and epigenetic alterations and molecular targets for therapeutic intervention for ALL, conventional chemotherapy is still the most important therapeutic approach. Relapses and high morbidity and mortality remain major challenges particularly in adult patients with ALL. Therefore, development of novel chemotherapeutic agents remains in demand for ALL patients. In the course of seeking novel agents against ALL, we screened a library of small molecules and identified that PQJS380, a S-(E)-4-([7S,10S]-4-ethyl-7-isopropyl-2,5,8,12-tetraoxo-9-oxa-3,6,13,18-tetraaza-bicycle[13,2,1] octadec-1-en-10-yl)but-3-enyl octanethioate, showed potent anti-leukemia activity. PQJS380 inhibited the proliferation with IC50 values of 14.25 nM and 5 nM in REH and NALM-6 cells, respectively. PQJS380 had 10-fold higher molar potency than the front-line ALL drugs Ara-C and VP-16. The median IC50 value for leukemia blast cells from 17 patients with ALL was 52 nM. PQJS380 induced G1-phase arrest in REH cells, and S-phase in NALM-6 cells, respectively. Treatment of PQJS380 led to apoptosis in ALL cell lines (REH and NALM-6) and primary ALL cells. Our data supported that PQJS380 may be a promising lead compound for ALL treatment even though the precise targets remain to be elucidated.

The Targeted Histone Deacetylase Inhibitor Tefinostat (CHR-2845) Shows Selective In Vitro Efficacy In Monocytoid-Lineage Acute Myeloid Leukaemia (AML)

Tefinostat (CHR-2845) is a novel monocyte/macrophage-targeted histone deacetylase inhibitor (HDACi) that is cleaved to an active acid, CHR-2847, by an intracellular esterase (human carboxylesterase-1, hCE-1), found only in cells of monocytoid lineage and hepatocytes. The clinical uptake of HDAC inhibition to date has been restricted by systemic toxicities including gastrointestinal disturbance, thrombocytopenia and fatigue. Accumulation of CHR-2847 in hCE-1-expressing cells results in a 20-100-fold increase in targeted anti-proliferative potency, considerably widening the potential therapeutic window in malignancies involving cells of monocytoid lineage (AML-M4, AML-M5 and CMML) by sparing the systemic toxicological effects associated with non-selective HDAC inhibition.The in vitro efficacy of tefinostat was assessed in primary AMLs using stored mononuclear cells obtained at diagnosis from 70 AML patients. Dose-dependent induction of apoptosis and significant growth inhibitory effects were seen in M4 /M5 AMLs (median IC50; 1.1µM+/-1.8) compared to non-M4/M5 FAB types (median IC50 5.1µM +/-4.7) (p=0.007). This potency and monocytoid specificity was not reproduced when using an alternative HDACi, tefinostat analogue CHR-8185 which is not cleaved by hCE-1.hCE-1 protein expression in patient samples was measured by both intracellular flow cytometry and immunoblotting, with highest levels seen in M4/M5 patients. This observation was validated by microarray analysis of hCE-1 mRNA in a further 130 AML samples with M4/M5 AMLs showing significant overexpression compared to normal bone marrow CD34+ cells (p=0.009). High levels of hCE-1 expression were found to drive a significant increase in tefinostat efficacy as measured by growth inhibition assays (p=0.001), and also strongly correlated with expression of the mature monocytoid marker CD14+.Sub-population analysis by flow cytometry revealed variable sensitivity to tefinostat within AML blasts, with CD14+ expressing cells showing maximum growth inhibition. This CD14+ response was accompanied by an induction of intracellular protein acetylation at nanomolar concentrations in tefinostat-responsive samples. Tefinostat-sensitive samples also showed strong induction of the cell cycle arrest and DNA damage sensor protein pH2AX, which is a potential biomarker of patient responsiveness.Importantly, no growth inhibitory effects were seen in normal bone marrow cells (n=5) exposed to AML-toxic doses of tefinostat while, in comparison, equivalent concentrations of the non-hCE-1-dependent analogue CHR-8185 caused considerable cytotoxicity, again emphasising the potential for expansion of the clinical therapeutic window using an hCE-1-dependent agent.In vitro synergy was demonstrated in combination experiments with tefinostat and cytarabine (median Combination Index value=0.68) which is likely to be a logical combination for future clinical evaluation.In summary, monocytoid targeting of HDACi activity was achieved using tefinostat in primary AML samples of monocytoid lineage, with minimal toxicity to normal bone marrow cells at equimolar concentrations. Given the absence of significant toxicity seen in a recently-published phase 1 study of tefinostat in patients with advanced haematological malignancies, further larger scale clinical evaluation of this compound is warranted in haematological malignancies involving cells of monocytoid lineage. Disclosures:Zabkiewicz:Chroma Therapeutics: Research Funding. Gilmour:Chroma Therapeutics: Research Funding. Hills:Chroma Therapeutics: Research Funding. Bone:Chroma Therapeutics: Employment. Davidson:Chroma Therapeutics: Employment. Burnett:Chroma Therapeutics: Research Funding. Knapper:Chroma Therapeutics: Research Funding.

Leukemia Stem Cell Marker CD123 (IL-3R alpha) Predicts Minimal Residual Disease and Relapse, Providing a Valid Target For SL-101 In Acute Myeloid Leukemia With FLT3-ITD Mutations

Leukemia stem cells (LSC) play critical role in initiation and progression of acute myeloid leukemia (AML). CD123 (IL-3R alpha) is one of the established LSC markers, and the frequency of CD123+ LSCs at diagnosis is associated with response to treatment (Vergez F et al, Haematologica 2011). However, it remains unclear whether CD123+ LSCs contribute to minimal residual disease (MRD) and disease recurrence.In this study, we first analyzed expression of CD123 on CD34+ blast cells in a cohort of 95 newly diagnosed AML patients (pts) treated with induction regimens between 02/2011 and 10/2012. In these pts, standard MRD assessment was routinely performed at the time of complete remission (CR) as described (Ravandi et al, Leukemia 2012). CD123 was highly expressed in diagnostic BM samples (67%, range 26.8%-91.7%). Higher expression of CD123+ cells on blasts and within CD34+ stem/progenitor cells correlated with poor prognosis cytogenetics (p=0.016 and 0.034) and unmutated NPM1 (p<0.001 and p=0.003, respectively). Altogether, 62.1% (59/95) pts achieved negative MRD, which in a multivariate analysis strongly correlated with longer time to relapse (HR: 0.71; 95%CI: 0.58-0.87; p<0.001). A higher proportion of CD123+CD34+ cells in remission marrows (ratio of %CD34+CD123+/%CD34+ greater than 0.83) was associated with higher risk to remain MRD-positive in both univariate (OR, 0.11-0.96, p=0.043) and multivariate Logistic Regression models (p=0.027). Although higher frequencies of CD34+CD123+ blasts significantly correlated with shorter time to relapse in the univariate Cox proportional hazard model (p=0.037), only white blood count, FLT3-ITD mutation and time to achieve MRD remained significant in a multivariate analysis.We next analyzed expression of CD123 in enriched CD34+CD38-CD99+ LSC fraction (Jan et al, Sci Transl Med. 2012) in 10 newly diagnosed AML BM samples. As controls, we used 3 normal bone marrow (NBM) samples. Cell surface characteristics and signaling pathways activation were defined by the time-of-flight mass cytometry (CyTOF) (Bendall et al, Science. 2011) using 11 cell surface markers (CD34, CD38, CD123, CD99, CD45, CD33, CD117, CD7, CD4, CD90 and CD133) and 8 intracellular markers (p-4EBP1, p-NF¦ÊB, p-STAT3, p-AKT, p-mTOR, p-ERK, p-S6 and p-STAT5). The frequency of CD123+ cells within CD34+CD38-CD99+ LSCs was significantly higher in AML samples at diagnosis (median 78.1%, range 38.4%-96.3%) compared to NBM (median 25.1%, range 22.8%-39.6%; p<0.001). FLT3-mutated AML expressed higher levels of CD123 within LSCs (median 90.9%, range 72.9%-96.3%) compared to FLT3-wt (median 69.5%, range 38.4%-73.5%; p=0.013). Multiple intracellular signaling pathways were highly activated in CD123+ LSCs in AML with FLT3-ITD compared to NBM stem cells, including p-4EBP1, p-NF¦ÊB, p-AKT, p-mTOR and p-ERK (Figure 1). This data indicate that targeting CD123-expressing LSC may have anti-leukemia effects in AML, especially those with FLT3 mutations. [Display omitted] To this end, we tested anti-leukemia activity of SL-101, a novel targeted therapy directed to CD123, in AML cell lines. SL-101 is an anti-CD123 scFv antibody conjugate. CD123 is highly expressed in AML cell lines (n=12) except for U937 and HL-60 cells (median 75.2%, range 0.9%-99.8%). SL101-induced killing strongly correlated with CD123 expression (r=-0.8220, p=0.001), suggesting that SL101 induces target-specific cell killing at low nanomolar range (median IC50 10.9 nM, range 0.1 nM-249.9 nM). We next analyzed the combined effects of SL101 and FLT3 inhibitor sorafenib in three FLT3-ITD cell lines. Strikingly, exogenous IL-3 significantly protected FLT3 mutant cells from sorafenib-induced cell killing in MV4;11 (p<0.01) and MOLM13 cells (p<0.001) but not in MOLM14 cells. In turn, combination with SL-101 revoked IL-3 mediated cytoprotection in MV4;11 (p<0.001) and MOLM13 cells (p<0.01).In conclusion, expression of CD123 on CD34+ blast cells is associated with persistent MRD in newly diagnosed AML. Multiple signaling pathways are activated in these CD123+ LSCs, supporting the feasibility of targeting CD123 to eliminate AML LSCs. SL-101 induces apoptotic cell death in CD123+ AML cell lines at low nanomolar concentrations and abrogates IL-3 mediated cytoprotection in FLT3-mutant cells. Ongoing studies are focusing on the anti-leukemia efficacy of SL-101 in primitive LSCs in primary AML samples, especially those with FLT3-ITD. Disclosures:Brooks:Stemline Therapeutics: Employment, Equity Ownership. Rowinsky:Stemline Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

The Survivin Suppressant YM155 (Sepantronium Bromide) Has Potent Pre-Clinical Activity Against Pediatric Acute Myeloid Leukemia

Despite aggressive chemotherapy and a high remission induction rate, approximately one third of children with acute myeloid leukemia (AML) relapse. Therefore, novel and more effective treatments are urgently needed. Survivin is an inhibitor-of-apoptosis protein that plays a key role in regulating cell division, proliferation and apoptosis (Altieri et al, Nat Rev Cancer, 2008). Furthermore, high expression of survivin and its splice variants have been shown to be associated with poor clinical outcome in AML (Moore et al, ASH Abstract 3555, 2011; Carter et al, Blood, 2012). The small-molecule survivin suppressant YM155 (sepantronium bromide) has been demonstrated to have pre-clinical activity against a range of solid cancers and leukemias, although data in AML is limited, particularly in pediatric models. Therefore, we undertook a comprehensive pre-clinical evaluation of YM155 in AML, with a focus on pediatric disease.When tested in vitro against a diverse panel of 9 AML cell lines, YM155 potently inhibited cell viability with a median IC50 of 0.03 µM (range 0.001 - 0.680 µM; Table 1). All 4 pediatric cell lines tested (Kasumi-1, MV4-11, THP-1 and CMK) were particularly sensitive to YM155, with IC50 values in the range of 0.010 - 0.053 µM. Of note, YM155 had generally similar in vitro efficacy to daunorubicin, but was more potent than cytarabine in 7 of the 9 cell lines.Table 1In vitro sensitivity of AML cell lines to YM155, cytarabine and daunorubicin, as measured by 72 hour cell viability (resazurin reduction) assays. Values indicate median IC50(µM) from at least 3 independent experiments. *Pediatric AML cell lines. NS, not significant.AML Cell LineCharacteristic FeatureYM155CytarabineDaunorubicinP value (YM155 vs. Cytarabine)HL-60Complex karyotype0.0010.4810.019NSKasumi-1*t(8;21)(q22;q22)0.0100.1120.0220.011RUNX1-CBFA2T1c-KITmut(N822K)ML-2t(6;11)(q27;q23)0.0100.0620.0050.027OCI/AML3NPM1mut0.011>100.020<0.001MV4-11*t(4;11)(q21;q23)0.0301.2630.004<0.001MLL-AF4FLT3-ITDTHP-1*t(9;11)(p21;q23)0.0367.0440.027<0.001CMK*AML assoc. with Down Syndrome0.0530.4280.078<0.001HELAML M60.5310.0780.1310.011ME-1inv(16)(p13q22)0.6800.3900.298NSCBFB-MYH11Cell cycle analyses demonstrated concentration-dependent increases in the sub-G1 fraction of cell lines treated with YM155, suggestive of apoptosis. An apoptotic mechanism of cell death was confirmed by an increase in annexin V positivity, measured by flow cytometry. Consistent with the proposed mechanism of action, YM155 treatment also caused down-regulation of total survivin protein expression in AML cell lines.Furthermore, in vitro assays demonstrated that YM155 was cytotoxic against a panel of 7 primary, diagnostic bone marrow samples from children with AML (median age 4.8 years, range 1.3-15.7). Patient samples were obtained from the Queensland Children's Tumour Bank and had diverse cytogenetic and molecular characteristics, including CBF-AML, monosomy 7, MLL gene rearrangements and FLT3-ITD. Similar to experiments in established cell lines, YM155 treatment caused an apoptotic response, as evidenced by induction of annexin V positivity.In conclusion, YM155 has potent pre-clinical activity against a broad panel of AML cell lines and patient samples. Pediatric AML appears to be particularly sensitive to YM155. These data suggest that YM155-mediated inhibition of survivin is a potentially beneficial therapeutic strategy for AML, particularly childhood disease, and warrants further pre-clinical and clinical evaluation. Disclosures:No relevant conflicts of interest to declare.

Selective Bcl-2 Inhibition With ABT-199 Is Highly Active Against Chronic Lymphocytic Leukemia (CLL) Irrespective Of TP53 Mutation Or Dysfunction

AimCLL is characterized by universal overexpression of the anti-apoptotic protein Bcl-2 which promotes inappropriate survival and chemotherapy resistance. ABT-199 is a Bcl-2 selective BH3-mimetic molecule currently in phase I clinical trials for the treatment of CLL. Del(17p) is a key negative prognostic indicator in CLL, associated with reduced response rates, progression-free and overall survival with standard therapy. Reduction or loss of TP53 function is considered the biological basis for its impact. Consistent with this, TP53 mutations in CLL lacking del(17p) also confer similar negative prognostic effects. BH3-mimetics are small molecule inhibitors that theoretically act downstream of TP53. We therefore hypothesized that ABT-199 will be equally effective in patients (pts) with CLL irrespective of TP53 function. We aimed to: (i) examine the incidence of TP53 aberrations in a heavily pretreated group of patients referred to two centers for ABT-199 therapy; (ii) determine the impact of TP53 aberrations on in vitro and clinical responses of CLL to ABT-199; and (iii) determine if CLL cells deficient in TP53 pathway function are as sensitive to ABT-199 as CLL with intact TP53 function. MethodsThis work was conducted in parallel with phase I clinical trials of ABT-199 monotherapy (NCT01328626; n = 25) or monotherapy followed by combination with rituximab (NCT01682616; n = 5) in pts with relapsed and refractory (r/r) CLL, after written, informed consent. Pts were tested for potential TP53 aberration using (i) FISH for del(17p); (ii) bone marrow (BM) immunohistochemistry (IHC) for elevated TP53 expression (>20% CLL cells staining positive on BM trephine); (iii) a 72hour in vitro nutlin-3a cytotoxicity assay for functional assessment of the TP53 pathway; and (iv) targeted next generation sequencing of the TP53 gene (with confirmatory Sanger sequencing) to detect deleterious mutations. Del(17p) and TP53 status of CLL samples at screening was determined and correlated with in vitro responses to ABT-199 after 24 hours and objective clinical responses to therapy with ABT-199 as determined within the trial protocol using iwCLL criteria. ResultsIn this r/r group of CLL pts, 13/30 (43%) had evidence of del(17p) by FISH (range 9 - 90% of cells); 15/28 (54%) had evidence of TP53 mutation by sequencing; and 7/29 (24%) had TP53 overexpression by IHC, all of whom had TP53 mutations. For 27 pts with data for all three assays, ten had no evidence of TP53 abnormality. In the 10 samples with >20% del(17p) by FISH, deleterious TP53 mutations were detected in all. For the purposes of comparing responses to ABT-199 by TP53 status, three categories were identified: negative for both del(17p) and p53 mutation, positive for one only, and positive for both (table 1). CLL cells in all groups were similarly highly sensitive in vitro to ABT-199 (ANOVA p>0.05). The overall response rate was at least 87.5% in all groups. Given that CLL is characterized by heterogeneity for TP53 aberrations within individual patients, we selected four samples with high level del(17p) and TP53 mutations for assessment of TP53 function. All showed resistance to nutlin 3a-induced cytotoxicity (median IC50 12mM [95%CI 9 – 41mM]) when compared with CLL cells without del(17p) or TP53 mutations which were 15 – 30-fold more sensitive. No differences in vitro sensitivity to ABT-199 were detected when these nutlin-3a resistant samples were compared with cells lacking TP53 mutations or del(17p). ConclusionsDefects in the TP53 pathway are present in the majority of patients with heavily pretreated CLL; novel treatment for such patients must include activity against TP53 aberrant clones. CLL cells lacking TP53 pathway function have similar sensitivity to ABT-199 in vitro as do TP53 pathway intact cells, and clinical responses were observed in ≥87.5% of patients. These data suggest that ABT-199 should be evaluated alone and in combination in pts with TP53-aberrant lymphoid neoplasms. [Display omitted] Disclosures:Anderson:Abbvie: Research Funding. Off Label Use: ABT-199 is an unlicensed drug currently in phase I and II studies in patients with chronic lymphocytic leukemia. Seymour:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees. Huang:Genentech: Employee of Walter and Eliza Hall Institute which recieves commercial income related to ABT-199. Other, Research Funding; Abbvie: Research Funding. Roberts:Abbvie: Research Funding; Genentech: Employee Walter and Eliza Hall Institute which recieves commercial income related to ABT-199. Other, Research Funding.

Abstract C206: Pediatric Preclinical Testing Program (PPTP) evaluation of BMN 673, an inhibitor of Poly-ADP Ribose Polymerase (PARP), alone and with Temozolomide (TMZ).

Abstract Introduction: BMN 673 is a potent and selective inhibitor of PARP1/2. Inhibitors of PARP such as BMN 673 show clinical activity against cancers lacking homologous repair through mutations in BRCA1 and 2. For Ewing sarcoma (ES), the EWS/FLI1 chimeric transcription factor increases PARP expression, and PARP appears to facilitate EWS/FLI1 function. Two reports have indicated that ES cell lines are more sensitive to PARP inhibitors than most other cell lines. Methods: BMN 673 was evaluated as a single agent and in combination with TMZ against the 23 cell lines of the PPTP in vitro panel using 96 hour exposure. In single-agent studies, daily oral BMN 673 administration was tested against the PPTP solid tumor xenografts (SCID mice) and acute lymphoblastic leukemia (ALL; NOD-SCID mice) panels using a dose of 0.16 mg/kg BID x 5 and 0.33 mg/kg QD on weekends for up to 28 days. In combination studies, two dose levels of BMN 673 (0.25 &amp; 0.1 mg/kg BID x 5 days) given with TMZ (12 &amp; 30 mg/kg/day x 5 days, respectively) were evaluated against ES xenografts. Standard PPTP measures of in vivo antitumor activity were employed to assess response. Results: The median relative IC50 (rIC50) for BMN 673 against the PPTP cell lines was 28 nM, with a range from 4 nM to &amp;gt;1000 nM. There was a trend for lower rIC50 values for the ES cell line panel. In combination with a fixed concentration of BMN 673 (10 nM), the TMZ rIC50 was markedly reduced for some PPTP cell lines, with ES cell lines showing up to a 60-fold reduction. In vivo, BMN 673 was well tolerated with only a 1.9% toxicity rate in the treated groups. BMN 673 induced significant improvements in event-free survival (EFS) distribution compared to control in 18 of 35 (51%) of the evaluable solid tumor xenografts, but in 0 of 8 ALL models. Only 2 of 34 (6%) evaluable solid tumor xenografts and no ALL models showed EFS T/C values &amp;gt; 2. Complete responses (CR) were observed for a Wilms tumor and a medulloblastoma model. BMN 673 in combination with TMZ induced CRs that were maintained through week 12 for 2 of 4 ES xenografts (TMZ dosed at either 12 or 30 mg/kg/day). Among an additional 6 ES xenografts that have recently initiated testing, 3 have ongoing CRs to the combination at Weeks 4 to 5. None of the ES xenografts responded to 5 days of treatment with single agent TMZ (30 mg/kg) or BMN 673 (0.5 mg/kg/day). Conclusions: Single agent BMN 673 shows limited activity against the PPTP solid tumor and ALL models, with no single agent activity against ES xenografts in vivo, despite ES cell lines demonstrating differential sensitivity in vitro. BMN 673 as a single agent induced CRs in 2 of 43 models, both of which are also highly responsive to cisplatin. Dramatic activity for the BMN 673 plus TMZ combination was observed for 5 of 10 ES models, with maintained CRs noted in vivo at TMZ doses as low as 12 mg/kg. Based on these results a pediatric phase 1 trial of BMN 673 plus TMZ is planned. (Supported by NO1-CM-42216) Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C206. Citation Format: Malcolm Smith, Min Kang, Patrick Reynolds, Richard Gorlick, Anders Kolb, John Maris, Richard Lock, Hernan Carol, Stephen Keir, Catherine Billups, Raushan Kurmasheva, Peter Houghton. Pediatric Preclinical Testing Program (PPTP) evaluation of BMN 673, an inhibitor of Poly-ADP Ribose Polymerase (PARP), alone and with Temozolomide (TMZ). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C206.

Abstract A129: Novel histone deacetylase inhibitors ITF-A and ITF-B exhibit efficacy in preclinical models of mature B-cell lymphomas.

Abstract Lymphomas are among the most common cancers. Despite significant progress made in their treatment, many patients still succumb to disease progression or relapse, or are refractory to current treatment regimens. Recently, the important role of epigenetic pathways in the pathogenesis of lymphomas has been highlighted, with several genes encoding epigenetic modifiers being shown to be selectively and prominently targeted by mutations and genomic aberrations. Histone deacetylase inhibitors (HDACi) are a class of molecules that have shown clinical efficacy in hematological malignancies, including T-cell lymphomas. Here, we have assessed the activity of two novel, class I, IIb, and IV HDACi, ITF-A and ITF-B, from Italfarmaco, in a panel of 29 non-Hodgkin lymphoma cell lines, comprising 21 diffuse large B-cell lymphoma (DLBCL) cell lines, 5 mantle cell lymphoma (MCL) cell lines, and 3 splenic marginal zone lymphoma (SMZL) cell lines. Cells were exposed to increasing concentrations of each HDACi and the MTT assay was performed after 72 hours to determine their cytotoxic and cytostatic effects estimating the IC50 (50%-inhibitory concentration), GI50 (50%-growth inhibition), LC50 (50%-lethal concentration), and TGI (total growth inhibition) values. Both ITF-A and ITF-B showed potent activities in most of the cell lines tested, irrespective of histological type, with the majority of IC50 values being below 100 nM. The median IC50 values were 13 nM (2-103 nM) and 33 nM (5-305 nM), for ITF-B and ITF-A, respectively. Median GI50 was 10 nM (1-63) for ITF-B, and 28 nM (2-217 nM) for ITF-A. The median LC50 values were 117 nM (10-2057 nM) and 42 nM (3-2388 nM) for ITF-A and ITF-B, respectively. The range of TGI values was 5-649 nM with a median of 59 nM for ITF-A, and 2-388 nM, with a median of 20n M for ITF-B. Both ITF-A and ITF-B induced apoptosis in the DLBCL and MCL tested as assessed by Annexin-V/7-AAD staining. Two DLBCL cell lines showing sensitivity to both HDACi, were chosen for further functional studies: DOHH2, a GCB-DLBCL cell line, and TMD8, a non-GCB DLBCL cell line. ITF-A and ITF-B presented both nuclear and cytoplasmic activity, as demonstrated by Western blotting analysis showing increased levels of both acetylated histone H3 and acetyl-α-tubulin following treatment with the two HDACi. Quantitative real-time PCR following 24 hours treatment of both cell lines with the two HDACi showed changes in the expression levels of STAT3, CDKN1A (p21) and MYC, suggesting roles for these genes in mediating the anti-proliferative effects of the two molecules. In conclusion, ITF-A and ITF-B showed promising activity in preclinical models of mature lymphomas and represent two new HDACi worthy of further pre-clinical evaluation as single agents and in combination with other agents. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A129. Citation Format: Afua A. Mensah, Eugenio Gaudio, Ivo Kwee, Andrea Rinaldi, Gianluca Fossati, Anastasios Stathis, Emanuele Zucca, Gianluca Caprini, Francesco Bertoni. Novel histone deacetylase inhibitors ITF-A and ITF-B exhibit efficacy in preclinical models of mature B-cell lymphomas. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A129.

Initial testing (stage 1) of the topoisomerase II inhibitor pixantrone, by the pediatric preclinical testing program

Pixantrone, a novel aza-anthracenedione with cytotoxic activity, was tested against the PPTP in vitro panel (3.0 nM to 30.0 μM) and against a limited panel of PPTP Wilms tumors and sarcomas (7.5 mg/kg) administered intravenously using an every 4 day × 3 schedule. In vitro pixantrone showed a median relative IC50 value of 54 nM (range <3 nM to 1.03 μM). In vivo pixantrone induced significant differences in EFS distribution compared to controls in two of eight solid tumor xenografts at dose levels relevant to human drug exposure. A complete response was observed for one Wilms tumor xenograft.

Initial testing (stage 1) of the histone deacetylase inhibitor, quisinostat (JNJ-26481585), by the Pediatric Preclinical Testing Program

Quisinostat (JNJ-26481585) is a second-generation pyrimidyl-hydroxamic acid histone deacetylase (HDAC) inhibitor with high cellular potency towards Class I and II HDACs. Quisinostat was selected for clinical development as it showed prolonged pharmacodynamic effects in vivo and demonstrated improved single agent antitumoral efficacy compared to other analogs. Quisinostat was tested against the PPTP in vitro panel at concentrations ranging from 1.0 nM to 10 μM and was tested against the PPTP in vivo panels at a dose of 5 mg/kg (solid tumors) or 2.5 mg/kg (ALL models) administered intraperitoneally daily × 21. In vitro quisinostat demonstrated potent cytotoxic activity, with T/C% values approaching 0% for all of the cell lines at the highest concentration tested. The median relative IC50 value for the PPTP cell lines was 2.2 nM (range <1-19 nM). quisinostat induced significant differences in EFS distribution compared to control in 21 of 33 (64%) of the evaluable solid tumor xenografts and in 4 of 8 (50%) of the evaluable ALL xenografts. An objective response was observed in 1 of 33 solid tumor xenografts while for the ALL panel, two xenografts achieved complete response (CR) or maintained CR, and a third ALL xenograft achieved stable disease. Quisinostat demonstrated broad activity in vitro, and retarded growth in the majority of solid tumor xenografts studied. The most consistent in vivo activity signals observed were for the glioblastoma xenografts and T-cell ALL xenografts.

Preclinical evaluation of the PARP inhibitor, olaparib, in combination with cytotoxic chemotherapy in pediatric solid tumors

Poly(ADP-ribose) polymerase (PARP) signals DNA damage and facilitates DNA repair. PARP inhibitors are being evaluated in cancers with defective DNA repair mechanisms or in combination with cytotoxic therapy or radiation. We evaluated the PARP inhibitor, olaparib, in combination with chemotherapy using in vitro and in vivo pediatric solid tumor models. The IC50 of olaparib alone and in combination with cytotoxic agents was determined in 10 pediatric solid tumor cell lines. Synergy was assessed using the combination index of Chou-Talalay. Olaparib alone and in combination with topotecan/cyclophosphamide was evaluated in xenograft models of Ewing sarcoma (RD-ES) and neuroblastoma (NGP). PAR activity was evaluated in cell lines and tumor lysates. Olaparib induced growth inhibition, median (range) IC50 = 3.6 (1-33.8) µM, and inhibited PAR activity in pediatric solid tumor cell lines. The addition of olaparib to DNA damaging agents resulted in additive to synergistic interactions. In RD-ES and NGP xenografts, olaparib inhibited PAR activity by 88-100% as a single agent and 100% when administered with cyclophosphamide/topotecan. Although the addition of olaparib did not antagonize the activity of cyclophosphamide/topotecan, clear evidence of synergy could not be demonstrated. In pediatric solid tumor cell lines, clinically achievable concentrations of single agent olaparib caused growth inhibition. Although the in vitro data demonstrated synergistic efficacy of olaparib when added to the camptothecins and alkylating agents, synergy was not discernible in vivo. Clinical trials of PARP inhibitors in combination DNA damaging agents are necessary to establish the role of PARP inhibitors in childhood cancer.

Initial testing (stage 1) of the polo‐like kinase inhibitor volasertib (BI 6727), by the Pediatric Preclinical Testing Program

Volasertib (BI 6727) is a potent inhibitor of Polo-like kinase 1 (Plk1), that is overexpressed in several childhood cancers and cell lines. Because of its novel mechanism of action, volasertib was evaluated through the PPTP. Volasertib was tested against the PPTP in vitro cell line panel at concentrations from 0.1 nM to 1.0 μM and against the PPTP in vivo xenograft panels administered IV at a dose of 30 mg/kg (solid tumors) or 15 mg/kg (ALL models) using a q7dx3 schedule. In vitro volasertib demonstrated cytotoxic activity, with a median relative IC50 value of 14.1 nM, (range 6.0-135 nM). Volasertib induced significant differences in EFS in 19 of 32 (59%) of the evaluable solid tumor xenografts and in 2 of 4 (50%) of the evaluable ALL xenografts. Volasertib induced tumor growth inhibition meeting criteria for intermediate EFS T/C (>2) activity in 11 of 30 (37%) evaluable solid tumor xenografts, including neuroblastoma (4 of 6) and glioblastoma (2 of 3) panels, and 2 of 4 ALL models. Objective responses (CR's) were observed for 4 of 32 solid tumor (two neuroblastoma, one glioblastoma, and one rhabdomyosarcoma) and one of four ALL xenografts. Volasertib shows potent in vitro activity against the PPTP cell lines with no histotype selectivity. In vivo, volasertib induced regressions in several xenograft models. However, pharmacokinetic data suggest that mice tolerate higher systemic exposure to volasertib than humans, suggesting that the current results may over-estimate potential clinical efficacy against the childhood cancers studied.

Substrate-Dependent Ligand Inhibition of the Human Organic Cation Transporter OCT2

Organic cation transporter 2 (OCT2) mediates the initial step in renal secretion of organic cations: uptake from the blood, across the basolateral membrane, and into the renal proximal tubule cells. Because of its potential as a target for unwanted drug-drug interactions (DDIs), considerable attention has been directed toward understanding the basis of OCT2 selectivity. These studies typically assess selectivity based on ligand inhibition profiles for OCT2-mediated transport of a probe substrate. However, little attention has been given to the potential influence of the substrate on the profile of ligand inhibition. Here we compared the IC50 values obtained for a set of structurally distinct inhibitors against OCT2-mediated transport of three structurally distinct substrates: 1-methyl-4-phenylpyridinium (MPP); metformin; and a novel fluorescent substrate, N,N,N-trimethyl-2-[methyl(7-nitrobenzo[c][l,2,5]oxadiazol-4-yl)amino]ethanaminium iodide (NBD-MTMA). The median IC50 value for inhibition of MPP transport was 9-fold higher than that for inhibition of metformin transport. Similarly, the median IC50 value for inhibition of MPP transport was 5-fold higher than that for NBD-MTMA transport. However, this was not a systematic difference in inhibitory efficacy; the ratio of IC50 values, MPP versus NBD-MTMA, ranged from 88-fold (ipratropium) to 0.3-fold (metformin). These data show that 1) the choice of OCT2 substrate significantly influences both quantitative and qualitative inhibitory interactions with cationic drugs; and 2) ligand interactions with OCT2 are not restricted to competition for a common ligand binding site, consistent with a binding surface characterized by multiple, possibly overlapping interaction sites. Development of predictive models of DDIs with OCT2 must take into account the substrate dependence of ligand interaction with this protein.

The Role of Pfmdr1 and Pfcrt in Changing Chloroquine, Amodiaquine, Mefloquine and Lumefantrine Susceptibility in Western-Kenya P. falciparum Samples during 2008–2011

Single Nucleotide Polymorphisms (SNPs) in the Pfmdr1, and Pfcrt, genes of Plasmodium falciparum may confer resistance to a number of anti-malaria drugs. Pfmdr1 86Y and haplotypes at Pfcrt 72-76 have been linked to chloroquine (CQ) as well as amodiaquine (AQ) resistance. mefloquine (MQ) and lumefantrine (LU) sensitivities are linked to Pfmdr1 86Y. Additionally, Pfcrt K76 allele carrying parasites have shown tolerance to LU. We investigated the association between Pfmdr1 86/Pfcrt 72-76 and P. falciparum resistance to CQ, AQ, MQ and LU using field samples collected during 2008–2011 from malaria endemic sites in western Kenya. Genomic DNA from these samples was genotyped to examine SNPs and haplotypes in Pfmdr1 and Pfcrt respectively. Additionally, immediate ex vivo and in vitro drug sensitivity profiles were assessed using the malaria SYBR Green I fluorescence-based assay. We observed a rapid but steady percent increase in wild-type parasites with regard to both Pfmdr1 and Pfcrt between 2008 and 2011 (p<0.0001). Equally, a significant reciprocate decrease in AQ and CQ median IC50 values occurred (p<0.0001) during the same period. Thus, the data in this study point to a significantly rapid change in parasite response to AQ and CQ in the study period. This may be due to releasing of drug pressure on the parasite from reduced use of AQ in the face of increased Artemisinin (ART) Combination Therapy (ACT) administration following the intervention of the Global Fund in 2008. LU has been shown to select for 76K genotypes, thus the observed increase in 76K genotypes coupled with significant cross resistance between LU and MQ, may herald emergence of tolerance against both drugs in future.

Lack of correlation between the size of HIV proviral DNA reservoir and the level of immune activation in HIV-infected patients with a sustained undetectable HIV viral load for 10 years

BackgroundThe persistence of HIV residual replication in patients with an undetectable plasma viral load (pVL) may limit immune recovery and facilitate inflammation-induced comorbidities. ObjectiveThe objective was to evaluate any correlation between immune restoration and intracellular [IC] HIV-DNA in cART-treated patients with a sustained undetectable pVL. Study designThis retrospective cross sectional study included 62 patients with a median duration of undetectable pVL of 10.3 years. IC HIV DNA in peripheral mononuclear blood cells (PBMCs) and T cell subsets were measured at the last visit. pVL, CD4+ and CD8+ T cell counts were retrospectively collected from the onset of long-term inhibition by antiretroviral treatment. The patients were separated into two groups: 27 non-blippers (sustained pVL<threshold value during all the visits) and 35 blippers (≥1 episodes of pVL>threshold but <1000 copies/ml). The median pVL in blippers was 115 copies/ml. ResultsThe median IC HIV DNA rate was 34 copies/106 PBMCs (71% ≥20 copies/106 PBMCs) with no significant difference between the groups. The proportion of CD8+CD38+ and CD8+DR+ T cells was higher in blipper patients, but the difference was only significant for the CD8+DR+ marker (p=0.036). No correlation was found between markers of immune activation on CD4+ and CD8+ T cells and the IC HIV-DNA level. ConclusionNo relation was found between the size of HIV reservoirs and immune activation in patients with sustained undetectable pVL. Mechanisms of immune activation have to be better understood in order to define specific therapeutic interventions.

Broadly Neutralizing Antibody PGT121 Allosterically Modulates CD4 Binding via Recognition of the HIV-1 gp120 V3 Base and Multiple Surrounding Glycans

New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC50 less than 0.05 µg ml−1. Here, we show that three family members, PGT121, PGT122 and PGT123, have very similar crystal structures. A long 24-residue HCDR3 divides the antibody binding site into two functional surfaces, consisting of an open face, formed by the heavy chain CDRs, and an elongated face, formed by LCDR1, LCDR3 and the tip of the HCDR3. Alanine scanning mutagenesis of the antibody paratope reveals a crucial role in neutralization for residues on the elongated face, whereas the open face, which accommodates a complex biantennary glycan in the PGT121 structure, appears to play a more secondary role. Negative-stain EM reconstructions of an engineered recombinant Env gp140 trimer (SOSIP.664) reveal that PGT122 interacts with the gp120 outer domain at a more vertical angle with respect to the top surface of the spike than the previously characterized antibody PGT128, which is also dependent on the N332 glycan. We then used ITC and FACS to demonstrate that the PGT121 antibodies inhibit CD4 binding to gp120 despite the epitope being distal from the CD4 binding site. Together, these structural, functional and biophysical results suggest that the PGT121 antibodies may interfere with Env receptor engagement by an allosteric mechanism in which key structural elements, such as the V3 base, the N332 oligomannose glycan and surrounding glycans, including a putative V1/V2 complex biantennary glycan, are conformationally constrained.