Abstract: Myelodysplastic syndromes (MDS) are usually defined as clonal proliferations of pluripotent stem cells, which retain their capacity to differentiate but do so in an inefficient manner, so that mature cells in the peripheral blood are variably altered and reduced. Various approaches can be used to prove the existence of a clonal population of cells. Analysis of X inactivation-dependent methylation patterns in double heterozygous females has been used in MDS, and this approach showed clonal hematopoietic progenitors capable of granulocytic-monocytic differentiation in most cases. However, in a recent study (Blood2003;102:1211–6) clonality, defined as a clonal population accounting for 35% or more of total granulocytes, was confirmed in only one third of patients with refractory anemia. We studied X-chromosome inactivation patterns (XCIP) in 50 consecutive female patients with MDS. These included 18 RA, 6 RARS, 12 RCMD, 4 RCMD-RS, 2 MDS with del(5q), 4 RAEB-1 and 4 RAEB-2. The XCIP was established by analysis of the IDS gene expression and of DNA methylation at the HUMARA and PGK loci. In order to assess the clonality status of single hematopoietic lineages, the relative expression of HUMARA alleles in reticulocytes, granulocytes and platelets was evaluated by RT-PCR. Finally, telomere length, as a major determinant of the replicative lifespan of hematopoietic cells, was analyzed on peripheral blood granulocytes by flow-FISH (Dako Telomere PNA kit/FITC). XCIP and telomere length analyses were performed at the diagnosis (21 cases) or during the follow-up (29 cases, median time from diagnosis 10 months, range 1–230). XCIP were informative in 43 patients: 20 displayed clonal or ambiguous XCIP (46.5%), while 23 showed polyclonal XCIP (53.5%). Polyclonal XCIP were found in 20 of 36 informative MDS patients without excess blasts (55.5%), and 3 of 7 (42.9%) with excess blasts. Among MDS patients with polyclonal hematopoiesis, clonal cytogenetic abnormalities were found in 9 out of 17 informative cases, all of them showing a chimeric karyotype with normal and abnormal metaphases. Telomere length was significantly shorter in patients with monoclonal patterns than in patients with polyclonal hematopoiesis (P=.01). A preliminary study of XCIP by expression of HUMARA alleles showed that 6 patients with polyclonal XCIP on PMN had clonal XCIP on both reticulocytes and platelets. In brief, these data provide evidence that more than 50% of patients with low risk MDS had some polyclonal hematopoiesis. However, detection of karyotypic abnormalities in these patients confirmed the clonal nature of the disease, the myelodysplastic clones coexisting with residual normal hematopoiesis. Preliminary data indicate that the myelodysplastic clone may originate in a common erythroid-megakaryocytic progenitor. Finally, the telomere data suggest that monoclonal hematopoiesis is associated with telomere shortening.

Abstract: Current therapy of CML with tyrosine kinase inhibitors (TKIs) aims to achieve a Major Molecular Response to avoid the disease progression to blastic phase and, possibly, a Deep Molecular Response (DMR), to gain the opportunity for TKIs discontinuation and Treatment Free Remission (TFR). However, TFR strategies are not yet optimized, as well as the criteria for patients (pts) selection to TKI discontinuation are not well defined. The patient's CML prognostic score, the duration of TKI treatment and "stable" DMR before discontinuation and the levels of DMR are still matters of debate. Due to its intrinsic limitations, RT-qPCR cannot be considered as an optimal tool for a precise detection of minimal BCR-ABL1 transcript levels and DMR monitoring. The digital PCR (dPCR) has emerged as a more sensitive and accurate method to detect and monitor the BCR-ABL1 minimal residual disease (MRD). This study focused on the BCR-ABL1 RT-q/dPCR comparative monitoring in CML pts treated with TKIs and with durable DMR (≥ 2 years), as conventionally assessed by RT-qPCR, before the enrollment. The aim was to evaluate the reliability and the efficiency of dPCR, as compared to RT-qPCR, for a better identification of "stable" DMR and for a better selection of the candidates for treatment discontinuation. 118 CML pts with durable DMR were enrolled and 493 peripheral blood samples were comparatively analyzed by both RT-qPCR, according to the last International Guidelines, and dPCR (QS3D Digital PCR System). RT-qPCR results were assessed as BCR-ABL1 %IS. dPCR results were assessed as number of BCR-ABL1 copies/□l of reaction and the molecular response was categorized as □ 0.468 or 〈 0.468 BCR-ABL1 copies/□l according to the previously reported cut off (Bernardi S. et al, J Mol Biom Diagn, 2017). Starting from the first RTq/dPCR comparative assessment, the 118 pts were monitored every 3/4 months for at least 3 time points. Grouping the 118 pts according to the RT-qPCR molecular results at the enrolment, 50 cases (42%) resulted in the MR4.0group and 68 (58%) in the MR 4.5-MR5.0 group. No significant differences were observed between these 2 groups in terms of clinical and hematological characteristics. At the same time, the 118 pts were divided in 2 groups based on the comparative dPCR molecular results, according to the above mentioned cut-off value: 29 (25%) and 89 (75%) cases had dPCR values □ and □0.468 BCR-ABL1 copies/□l, respectively. No significant clinical differences were observed between these 2 groups. Out of these 118 pts, 87 (74%) discontinued the TKI treatment and 24 (28%) of them lost the DMR after a median time of 3 months (range 1-30). Based on dPCR, 23/87 (26%) and 64/87 (74%) had dPCR □ 0.468 and 〈 0.468, respectively. Considering the pts who lost DMR, 12/23 (41%) and 12/64 (20%), had dPCR □ 0.468 and 〈 0.468, respectively (p=0.0021). Therefore, evaluating BCR-ABL1 transcript levels by dPCR before the discontinuation, we could observe a better TFR (positive predictive value of 80%) in those pts with dPCR 〈 0.468, with respect to the ones with □ 0.468. However, no significant difference was observed between the groups divided according to the evaluation of BCR-ABL1 transcript levels by RT-qPCR before the discontinuation in terms of TFR prediction. In order to reach a better recognition of stable DMR, which is strictly related with the probability to achieve and maintain TFR after TKIs discontinuation, we comparatively analyzed, by RTq/dPCR, the course of MRD during the follow up period. In the case of monitoring by RT-qPCR, a wide oscillation of the molecular responses was observed and they tended to be superimposable both in the group of pts with MR4.0 and in those with MR4.5 and 5.0. In the case of monitoring by dPCR, the values assessed at the enrolment and during the follow up revealed the capability of the dPCR to measure different and heterogeneous numbers of BCR-ABL1 copies in each pts. Moreover, the pts with a dPCR below the cut-off value of 0.468, at enrolment, were maintaining values (median 0,175; range 0-1,863), significantly lower (p 〈 0.0001) than that (median 0,393; range 0-2,195) of the pts with starting values of dPCR above the cut-off of 0.468 (Fig 1). This retrospective study show that the dPCR is more accurate and sensitive than conventional RT-qPCR for detecting and monitoring the BCR-ABL1 molecular levels and potentially able to improve the recognition of the stable DMR and selection of the candidates to TKI treatment discontinuation. Figure 1. Figure 1. Disclosures Bonifacio: Incyte: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Novartis: Research Funding; Bristol Myers Squibb: Consultancy. Gaidano:Morphosys: Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Roche: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Rossi:Teva: Membership on an entity's Board of Directors or advisory committees; Mundipharma: Honoraria; Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees; Sandoz: Honoraria; Jazz: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; Amgen: Membership on an entity's Board of Directors or advisory committees. Gobbi:Novartis: Consultancy; Ariad: Membership on an entity's Board of Directors or advisory committees; Pfister: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Janssen: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees. Voso:Celgene: Research Funding, Speakers Bureau. Abruzzese:Ariad: Consultancy; BMS: Consultancy; Novartis: Consultancy; Pfizer: Consultancy.

Abstract: Abstract 736 In erythroid cells from patients with refractory anemia with ringed sideroblasts (RARS), the expression of mitochondrial ferritin (MtF) - encoded by the nuclear FTMT gene located on chromosome 5q21.3 - occurs at a very early stage of differentiation and is closely related to the sideroblastic phenotype [Blood. 2003 Mar 1;101(5):1996-2000]. On the other hand, in extra-hematopoietic tissues MtF expression is associated with high cellular metabolic activity and oxygen consumption, suggesting a possible role of this protein in protecting mitochondria from iron-dependent oxidative damage. It is known that reactive oxygen species (ROS) act as second messengers in the JAK/STAT pathway, which is involved in the regulation of erythropoiesis, and that ROS scavengers inhibit STAT5 phosphorylation. It was the aim of this study to investigate the influence of experimentally induced MtF over-expression on erythroid differentiation in normal hematopoietic progenitors, and in addition to define the relationship between MtF expression and erythroid maturation, apoptosis, and JAK-STAT pathway activation in RARS. A liquid culture model was adopted to expand erythroid progenitors from CD34+ cells in the presence of IL-3, IL-6, stem cell factor and Epo [Blood. 2005 Jul 1;106(1):247-53] . To study the effect of MtF induction in normal hematopoiesis, CD34+ cells from 5 healthy donors were transduced using lentivirus carrying cDNA of FTMT downstream to the ubiquitous PGK promoter (transduction efficiency equal to 30-40%), and then cultured for 21 days. To assess the effect of MtF expression in myelodysplastic syndromes (MDS), CD34+ cells from 24 patients with RARS, as well as cells from 20 patients with refractory anemia (RA) and from 8 healthy donors (as control groups) were cultured. Cytospins were performed for MtF, H-ferritin (HF) and L-ferritin (LF) immunocytochemical analysis, and samples of cultured cells were removed at various days of culture for biological studies. Lentivirus-mediated FTMT transduction of normal CD34+ progenitors did not inhibit cell growth nor prevent the differentiation of erythroid progenitors. By flow cytometry analysis, MtF-positive erythroid progenitors showed significantly reduced levels of HF and increased expression of transferrin receptor (CD71) compared with MtF-negative progenitors (P = .004 and P = .01, respectively). In this model, induction of MtF resulted in increased cellular apoptosis (median number of apoptotic cells by TUNEL assay at day 21 equal to 83% in MtF-positive cells vs 18% in MtF-negative cells, P 〈 .001). A significantly lower proliferation rate and higher apoptotic index were observed in cultures from patients with RARS and RA with respect to healthy controls. FTMT mRNA was detected by RT-PCR in CD34+ progenitor cells from patients with RARS, while protein expression was observed only from day 4 of culture, with a significant increase in the percentage of MtF-positive cells during culture (median value from 5% at day 4 to 24% at day 21, P = .002). No MtF expression was observed in RA patients. In MtF-positive cells from RARS patients, an inverse relationship between MtF and HF cellular content, and a direct relationship between MtF and CD71 expression were observed. In erythroid progenitors from RARS patients, the apoptotic rate was higher in MtF-positive than in MtF-negative cells (median number of apoptotic cells equal to 17% vs 6%, P 〈 .001). Finally, we analyzed by flow cytometry the relationship between MtF expression and STAT5 phosphorylation in cultured cells following Epo stimulation. Preliminary data from three RARS patients showed that p-STAT5 expression was lower in MtF-positive than in MtF-negative cells (P = .02). In conclusion, experimental overexpression of MtF in normal erythroid progenitors may reduce mitochondrial iron availability thus inducing apoptosis. In erythroid progenitors from RARS patients, the pathological expression of MtF is associated with increased apoptosis of immature red cells (ineffective erythropoiesis), which may be at least in part determined by reduced activation of the JAK-STAT pathway in response to Epo. Disclosures: No relevant conflicts of interest to declare.

Abstract: Background Myelofibrosis (MF) is featured by an inflammatory condition that can also drive the progression of disease. Ruxolitinib (RUX) is the-first-in-class Jak1/2 inhibitor approved for treatment for MF. Clinical benefits of RUX are presumably derived from reduction of inflammatory cytokines even if the exact mechanism remains unclear. Recent reports have identified the ratio between absolute neutrophils count (ANC) and absolute lymphocyte count (ALC), called NLR, as a simple parameter that mirrors the inflammatory status and the myeloid associated immune suppression. In various malignancies NLR has been indicated as predictor of progression free survival (PFS) and overall survival (OS). Our preliminary work in a single-center experience showed that patients with NLR 〉 6 before RUX start had a lower chance to obtain 〉 50% spleen reduction in the first 12 weeks or a complete resolution of splenomegaly at 24 weeks. Objective : We proposed to test NLR=6 as bio-marker in MF to apply into clinical practice as a possible predictor of response to RUX. Methods We used two separate cohorts to validate NLR (as a continuous variable and as a cut off 6) as predictor of response to RUX bases on our preliminary data from healthy volunteers (data not shown). Cohort (#1) including 111 MF patients from MD Anderson Cancer Center treated with RUX on phase 1/2 clinical trial from 2007 to 2010; and cohort (#2) including 367 patients treated at 18 Italian centers between years 2012 - 2018. Spleen responses to RUX treatment, PFS and OS were independently validated in cohorts #1 and 2. As cohort 1 included patients treated on clinical trial, spleen was assessed by MRI before and after 24 weeks of RUX therapy, and by physical examination at week 12. In cohort #2, spleen size was assessed by physical examination before, after 12 and 24 weeks of RUX continuous treatment in a real-life setting. NLR was calculated using data obtained from the complete blood count before RUX start and correlated with driver mutations, early spleen reduction, progression free survival (PFS), defined as time from RUX start to last follow-up or progressive disease (including progression to acute myeloid leukemia, ≥20% blasts in peripheral blood or bone marrow, AML) or death for any reason; and overall survival (OS). Results : Clinical and demographics characteristics of patients in each cohort are summarized in Table 1. In cohort #1 we found that NLR was lower in patients with lower bone marrow fibrosis (grade 0-1: 6.2±0.8 versus grade 2-3: 7.3±0.8, p=0.03). Similarly, in cohort #2, patients with grade 0-1 bone marrow fibrosis had lower NLR than those carrying grade 2-3 bone marrow fibrosis (7.7±0.7 versus 10.6±1.3, p=0.04). NLR was higher in patients carrying JAK2 (V617F) mutation (mean +/- SD, 6.4±0.6 vs 5.3±0.5, p=0.02 in cohort 1 and 9.1±0.6 vs 5.0±0.5, p=0.002 in cohort 2). While in cohort 1 NLR appeared lower in CALR (exon 9 indel) mutated patients, the difference was statistically significant in cohort 2 (5.4±0.8 vs 8.9±0.6, p=0.03). In both cohorts, there were no differences in NLR in either triple negative or MPL (exon 10) patients. In cohort 1, the mean percentage change from baseline in palpable spleen length was −47.7% at week 12 and −53.4% at week 24. NLR=6 was able to identify at baseline early response to RUX with 66.9% sensitivity and 72.3% specificity (HR 1.68, p=0.01). Patients with NLR 〉 6 before RUX start had a lower chance to obtain a complete resolution of splenomegaly at 24 weeks (p=0.001). These observations were confirmed in cohort 2 where NLR 〉 6 was able to identify at baseline early response to RUX with 50.3% sensitivity and 67.7% specificity (HR 1.56, p=0.01). The mean percentage change from baseline in palpable spleen length was −60.3% at week 12 and −66.7% at week 24. Patients with NLR 〉 6 before RUX start had a lower chance to obtain a complete resolution of splenomegaly at 24 weeks (p 〈 0.002). At the time of this analysis, 84/111 (75.6%) patients in cohort 1 and 122/367 (33.2%) in cohort 2 had died (p 〈 0.001). Progression to AML occurred in 6/111 (5.4 %) patients of cohort 1 and in 35/367 (9.5%) patients of cohort 2 (p=0.03). With median follow-ups of 47.8 months and 35.2 months for cohorts 1 and 2, respectively, NLR as a continuous variable or NLR 〉 6 was not a predictor of PFS or OS. Conclusions : NLR before RUX start could serve as a useful, simple and early predictor of spleen response in MF patients; and it positively correlates with JAK2 mutation and higher fibrosis grade. Disclosures Palandri: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Vitolo:Sandoz: Speakers Bureau; Takeda: Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Speakers Bureau. Cuneo:Roche: Other: advisory board, Speakers Bureau; Abbvie: Other: advisory board, Speakers Bureau; Gilead: Other: advisory board, Speakers Bureau; janssen: Other: advisory board, Speakers Bureau. Aversa:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Basilea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria. Cavo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Palumbo:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Di Raimondo:Takeda: Honoraria, Research Funding; Celgene: Honoraria. Verstovsek:Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Italfarmaco: Membership on an entity's Board of Directors or advisory committees.

Abstract: The synthetic retinoid 6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437), which was originally developed as an retinoic acid receptor (RAR)-γ agonist, induces rapid apoptosis in all-trans retinoic acid (ATRA)-sensitive and ATRA-resistant clones of the NB4 cell line, a widely used experimental model of acute promyelocytic leukemia (APL). In addition, the compound is apoptogenic in primary cultures of freshly isolated APL blasts obtained from a newly diagnosed case and an ATRA-resistant relapsed patient. NB4 cells in the S-phase of the cycle are most sensitive to CD437-triggered apoptosis. CD437-dependent apoptosis does not require de novo protein synthesis and activation of RAR-γ or any of the other nuclear retinoic acid receptors. The process is preceded by rapid activation of a caspase-like enzymatic activity capable of cleaving the fluorogenic DEVD but not the fluorogenic YVAD tetrapeptide. Increased caspase activity correlates with caspase-3 and caspase-7 activation. Inhibition of caspases by z-VAD suppresses the nuclear DNA degradation observed in NB4 cells treated with CD437, as well as the degradation of pro–caspase-3 and pro–caspase-7. CD437-dependent activation of caspases is preceded by release of cytochrome c from the mitochondria into the cytosol of treated cells. Leakage of cytochrome c lays upstream of caspase activation, because the phenomenon is left unaffected by pretreatment of NB4 cells with z-VAD. Treatment of APL cells with CD437 is associated with a caspase-dependent degradation of promyelocytic leukemia-RAR-, which can be completely inhibited by z-VAD.

Abstract: Background. Ruxolitinib (RUX) is the first commercially available JAK1/2 inhibitor that may control splenomegaly and systemic symptoms related to myelofibrosis (MF). Despite MF occur frequently in elderly patients (pts), no data are yet available on RUX efficacy and safety in this particularly frail population. Methods We report on 100 pts [M/F 57/43, median age at diagnosis 75.7 years, interquartile range (IQR) 72.3 - 78.0, median age at baseline of RUX treatment 77.7 years, IQR 76.2 - 80.3] with WHO-defined MF treated with RUX when aged ≥ 75 years. Data were extracted from the whole cohort of 408 pts of any age collected in a database involving 22 Italian Centers. Comorbidities were recorded at the time of diagnosis and classified according to the Charlson Comorbidity Index (CCI). Response to RUX was evaluated according to IWG-MRT criteria. Results Main clinical features after stratification according to age at RUX start are reported in Table 1. Compared to younger pts, elderly pts carried a higher number of co-morbidities and had lower hemoglobin and platelet values, thus starting RUX with lower doses. Time from diagnosis to RUX start was comparable among the two cohorts (median 15.5 months, IQR 4.6 - 66.7, vs 20.8 months, IQR 4.1 - 66.0, p=0.74). According to IWG criteria, a spleen response was achieved by 37 out of 90 (41.1%) evaluable elderly pts compared to 115 out of 272 (42.2%) pts 〈 75y (p=0.85) while symptom response was achieved by 88/99 (88.8%) elderly pts compared to 271/304 (89.1%) younger pts (p=0.94). Drug-related anemia (Hb 〈 10 g/dl in pts with baseline Hb ≥10 g/dl) was observed in 30/68 (44.1%) evaluable elderly pts compared with 100/240 (41.6%) evaluable younger subjects (p=0.72). The percentage of pts that decreased RUX dose over time was comparable in the two groups (29% and 29.8%, respectively). Overall, 47% elderly and 32% younger pts finally discontinued RUX (p=0.008) after a median time of 12.3 and 21.6 months, respectively (p=0.03). Evolution into acute leukemia occurred in 8 (8.0%) elderly pts and in 22 (7.1%) younger pts, respectively (p=0.78), with a similar evolution-free survival from RUX initiation (p=0.35). As expected, 43 (43.0%) elderly pts and 53 (17.3%) younger pts died (p 〈 0.001) after a median time from RUX start of 14.2 and 24.2, respectively (p=0.03). Causes of death in elderly pts were: progression of myelofibrosis (32.5%), heart disease (16.3%), infections (14%), acute leukemia (7%), hemorrhage/thrombosis (7%), other unrelated causes (23.2%). Compared to elderly, younger pts died less frequently due to heart disease (3.6%) (p=0.03), and more frequently due to acute leukemia (23.2%) (p=0.03). The 4-year cumulative Event-Free Survival (taking into account: RUX discontinuation, blastic evolution and death for any cause) was 30.1% (95% CI: 16.2 - 44.0) in elderly pts and 46.1% (95% CI 37.3 - 54.9) in younger subjects, respectively (p=0.002). Conclusions. Despite the elderly carried a higher number of comorbidities and were treated with lower starting and titrated doses of RUX,RUX was feasible and effective in this setting, achieving clinical responses similar to younger subjects, with comparable toxicity rates. Thus, the study do not support to restrain the use of RUX based on older age and comorbidities. Figure Figure. Disclosures Latagliata: Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Janssen: Consultancy, Honoraria; Shire: Honoraria. Bonifacio:Ariad Pharmaceuticals: Consultancy; Amgen: Consultancy; Bristol Myers Squibb: Consultancy; Pfizer: Consultancy; Novartis: Research Funding. Tiribelli:Novartis: Consultancy, Speakers Bureau; Ariad Pharmaceuticals: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau. Cavo:Bristol-Myers Squibb: Honoraria; Amgen: Honoraria; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Celgene: Honoraria, Research Funding. Breccia:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Ariad: Honoraria; Pfizer: Honoraria.

Abstract: The synthetic retinoid 6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437), which was originally developed as an retinoic acid receptor (RAR)-γ agonist, induces rapid apoptosis in all-trans retinoic acid (ATRA)-sensitive and ATRA-resistant clones of the NB4 cell line, a widely used experimental model of acute promyelocytic leukemia (APL). In addition, the compound is apoptogenic in primary cultures of freshly isolated APL blasts obtained from a newly diagnosed case and an ATRA-resistant relapsed patient. NB4 cells in the S-phase of the cycle are most sensitive to CD437-triggered apoptosis. CD437-dependent apoptosis does not require de novo protein synthesis and activation of RAR-γ or any of the other nuclear retinoic acid receptors. The process is preceded by rapid activation of a caspase-like enzymatic activity capable of cleaving the fluorogenic DEVD but not the fluorogenic YVAD tetrapeptide. Increased caspase activity correlates with caspase-3 and caspase-7 activation. Inhibition of caspases by z-VAD suppresses the nuclear DNA degradation observed in NB4 cells treated with CD437, as well as the degradation of pro–caspase-3 and pro–caspase-7. CD437-dependent activation of caspases is preceded by release of cytochrome c from the mitochondria into the cytosol of treated cells. Leakage of cytochrome c lays upstream of caspase activation, because the phenomenon is left unaffected by pretreatment of NB4 cells with z-VAD. Treatment of APL cells with CD437 is associated with a caspase-dependent degradation of promyelocytic leukemia-RAR-, which can be completely inhibited by z-VAD.