HDAC1,2 inhibition and doxorubicin impair Mre11-dependent DNA repair and DISC to override BCR-ABL1-driven DSB repair in Philadelphia chromosome-positive B-cell precursor acute lymphoblastic leukemia.

Philadelphia chromosome-positive (Ph+) B-cell precursor acute lymphoblastic leukemia (ALL) expressing BCR-ABL1 oncoprotein is a major subclass of ALL with poor prognosis. BCR-ABL1-expressing leukemic cells are highly dependent on double-strand break (DSB) repair signals for their survival. Here we report that a first-in-class HDAC1,2 selective inhibitor and doxorubicin (a hyper-CVAD chemotherapy regimen component) impair DSB repair networks in Ph+ B-cell precursor ALL cells using common as well as distinct mechanisms. The HDAC1,2 inhibitor but not doxorubicin alters nucleosomal occupancy to impact chromatin structure, as revealed by MNase-Seq. Quantitative mass spectrometry of the chromatin proteome along with functional assays showed that the HDAC1,2 inhibitor and doxorubicin either alone or in combination impair the central hub of DNA repair, the Mre11-Rad51-DNA ligase 1 axis, involved in BCR-ABL1-specific DSB repair signaling in Ph+ B-cell precursor ALL cells. HDAC1,2 inhibitor and doxorubicin interfere with DISC (DNA damage-induced transcriptional silencing in cis)) or transcriptional silencing program in cis around DSB sites via chromatin remodeler-dependent and -independent mechanisms, respectively, to further impair DSB repair. HDAC1,2 inhibitor either alone or when combined with doxorubicin decreases leukemia burden in vivo in refractory Ph+ B-cell precursor ALL patient-derived xenograft mouse models. Overall, our novel mechanistic and preclinical studies together demonstrate that HDAC1,2 selective inhibition can overcome DSB repair 'addiction' and provide an effective therapeutic option for Ph+ B-cell precursor ALL.

BET protein proteolysis targeting chimera (PROTAC) exerts potent lethal activity against mantle cell lymphoma cells.

Bromodomain extraterminal protein (BETP) inhibitors transcriptionally repress oncoproteins and nuclear factor-κB (NF-κB) target genes that undermines the growth and survival of mantle cell lymphoma (MCL) cells. However, BET bromodomain inhibitor (BETi) treatment causes accumulation of BETPs, associated with reversible binding and incomplete inhibition of BRD4 that potentially compromises the activity of BETi in MCL cells. Unlike BETi, BET-PROTACs (proteolysis-targeting chimera) ARV-825 and ARV-771 (Arvinas, Inc.) recruit and utilize an E3-ubiquitin ligase to effectively degrade BETPs in MCL cells. BET-PROTACs induce more apoptosis than BETi of MCL cells, including those resistant to ibrutinib. BET-PROTAC treatment induced more perturbations in the mRNA and protein expressions than BETi, with depletion of c-Myc, CDK4, cyclin D1 and the NF-κB transcriptional targets Bcl-xL, XIAP and BTK, while inducing the levels of HEXIM1, NOXA and CDKN1A/p21. Treatment with ARV-771, which possesses superior pharmacological properties compared with ARV-825, inhibited the in vivo growth and induced greater survival improvement than the BETi OTX015 of immune-depleted mice engrafted with MCL cells. Cotreatment of ARV-771 with ibrutinib or the BCL2 antagonist venetoclax or CDK4/6 inhibitor palbociclib synergistically induced apoptosis of MCL cells. These studies highlight promising and superior preclinical activity of BET-PROTAC than BETi, requiring further in vivo evaluation of BET-PROTAC as a therapy for ibrutinib-sensitive or -resistant MCL.

Highly effective combination of LSD1 (KDM1A) antagonist and pan-histone deacetylase inhibitor against human AML cells.

This corrects the article DOI: 10.1038/leu.2014.119.

Novel BET protein proteolysis-targeting chimera exerts superior lethal activity than bromodomain inhibitor (BETi) against post-myeloproliferative neoplasm secondary (s) AML cells.

The PROTAC (proteolysis-targeting chimera) ARV-825 recruits bromodomain and extraterminal (BET) proteins to the E3 ubiquitin ligase cereblon, leading to degradation of BET proteins, including BRD4. Although the BET-protein inhibitor (BETi) OTX015 caused accumulation of BRD4, treatment with equimolar concentrations of ARV-825 caused sustained and profound depletion (>90%) of BRD4 and induced significantly more apoptosis in cultured and patient-derived (PD) CD34+ post-MPN sAML cells, while relatively sparing the CD34+ normal hematopoietic progenitor cells. RNA-Seq, Reverse Phase Protein Array and mass cytometry 'CyTOF' analyses demonstrated that ARV-825 caused greater perturbations in messenger RNA (mRNA) and protein expressions than OTX015 in sAML cells. Specifically, compared with OTX015, ARV-825 treatment caused more robust and sustained depletion of c-Myc, CDK4/6, JAK2, p-STAT3/5, PIM1 and Bcl-xL, while increasing the levels of p21 and p27. Compared with OTX015, PROTAC ARV-771 treatment caused greater reduction in leukemia burden and further improved survival of NSG mice engrafted with luciferase-expressing HEL92.1.7 cells. Co-treatment with ARV-825 and JAK inhibitor ruxolitinib was synergistically lethal against established and PD CD34+ sAML cells. Notably, ARV-825 induced high levels of apoptosis in the in vitro generated ruxolitinib-persister or ruxolitinib-resistant sAML cells. These findings strongly support the in vivo testing of the BRD4-PROTAC based combinations against post-MPN sAML.

Pre-clinical efficacy of combined therapy with novel ß-catenin antagonist BC2059 and histone deacetylase inhibitor against AML cells.

The canonical wingless-type MMTV integration site (WNT)-ß-catenin pathway is essential for self-renewal, growth and survival of acute myeloid leukemia (AML) stem/blast progenitor cells (BPCs). Deregulated WNT signaling inhibits degradation of ß-catenin, causing increased nuclear translocation and co-factor activity of ß-catenin with the transcriptional regulator T-cell factor (TCF) 4/lymphoid enhancer factor 1 in AML BPCs. Here, we determined the pre-clinical anti-AML activity of the anthraquinone oxime-analog BC2059 (BC), known to attenuate ß-catenin levels. BC treatment disrupted the binding of ß-catenin with the scaffold protein transducin ß-like 1 and proteasomal degradation and decline in the nuclear levels of ß-catenin. This was associated with reduced transcriptional activity of TCF4 and expression of its target genes, cyclin D1, c-MYC and survivin. BC treatment dose-dependently induced apoptosis of cultured and primary AML BPCs. Treatment with BC also significantly improved the median survival of immune-depleted mice engrafted with either cultured or primary AML BPCs, exhibiting nuclear expression of ß-catenin. Co-treatment with the pan-histone deacetylase inhibitor panobinostat and BC synergistically induced apoptosis of cultured and primary AML BPCs, including those expressing FLT3-ITD, as well as further significantly improved the survival of immune-depleted mice engrafted with primary AML BPCs. These findings underscore the promising pre-clinical activity and warrant further testing of BC against human AML, especially those expressing FLT3-ITD.

Highly effective combination of LSD1 (KDM1A) antagonist and pan-histone deacetylase inhibitor against human AML cells.

The histone demethylase LSD1 (KDM1A) demethylates mono- and di-methylated (Me2) lysine (K) 4 on histone H3. High LSD1 expression blocks differentiation and confers a poor prognosis in acute myeloid leukemia (AML). Here, treatment with the novel LSD1 antagonist SP2509 attenuated the binding of LSD1 with the corepressor CoREST, increased the permissive H3K4Me3 mark on the target gene promoters, and increased the levels of p21, p27 and CCAAT/enhancer binding protein α in cultured AML cells. In addition, SP2509 treatment or LSD1 shRNA inhibited the colony growth of AML cells. SP2509 also induced morphological features of differentiation in the cultured and primary AML blasts. SP2509 induced more apoptosis of AML cells expressing mutant NPM1 than mixed-lineage leukemia fusion oncoproteins. Treatment with SP2509 alone significantly improved the survival of immune-depleted mice following tail-vein infusion and engraftment of cultured or primary human AML cells. Co-treatment with pan-HDAC inhibitor (HDI) panobinostat (PS) and SP2509 was synergistically lethal against cultured and primary AML blasts. Compared with each agent alone, co-treatment with SP2509 and PS significantly improved the survival of the mice engrafted with the human AML cells, without exhibiting any toxicity. Collectively, these findings show that the combination of LSD1 antagonist and pan-HDI is a promising therapy warranting further testing against AML.

Phase Ia/II, two-arm, open-label, dose-escalation study of oral panobinostat administered via two dosing schedules in patients with advanced hematologic malignancies.

Panobinostat is a potent oral pandeacetylase inhibitor that leads to acetylation of intracellular proteins, inhibits cellular proliferation and induces apoptosis in leukemic cell lines. A phase Ia/II study was designed to determine the maximum-tolerated dose (MTD) of daily panobinostat, administered on two schedules: three times a week every week or every other week on a 28-day treatment cycle in patients with advanced hematologic malignancies. The criteria for hematologic dose-limiting toxicities differed between patients with indications associated with severe cytopenias at baseline (leukemia and myeloid disorders) and those less commonly associated with baseline cytopenias (lymphoma and myeloma). In patients with leukemia and myeloid disorders, 60 mg was the MTD for weekly as well as biweekly panobinostat. In patients with lymphoma and myeloma, 40 mg was the recommended dose for phase II evaluation (formal MTD not determined) of weekly panobinostat, and 60 mg was the MTD for biweekly panobinostat. Overall, panobinostat-related grade 3-4 adverse events included thrombocytopenia (41.5%), fatigue (21%) and neutropenia (21%). Single-agent activity was observed in several indications, including Hodgkin lymphoma and myelofibrosis. This phase Ia/II study provided a broad analysis of the safety profile and efficacy of single-agent panobinostat in patients with hematologic malignancies.

Prediction of outcomes in patients with Ph+ chronic myeloid leukemia in chronic phase treated with nilotinib after imatinib resistance/intolerance.

The purpose was to assess predictive factors for outcome in patients with chronic myeloid leukemia (CML) in chronic phase (CML-CP) treated with nilotinib after imatinib failure. Imatinib-resistant and -intolerant patients with CML-CP (n=321) were treated with nilotinib 400 mg twice daily. Of 19 baseline patient and disease characteristics and two response end points analyzed, 10 independent prognostic factors were associated with progression-free survival (PFS). In the multivariate analysis, major cytogenetic response (MCyR) within 12 months, baseline hemoglobin ≥ 120 g/l, baseline basophils <4%, and absence of baseline mutations with low sensitivity to nilotinib were associated with PFS. A prognostic score was created to stratify patients into five groups (best group: 0 of 3 unfavorable risk factors and MCyR by 12 months; worst group: 3 of 3 unfavorable risk factors and no MCyR by 12 months). Estimated 24-month PFS rates were 90%, 79%, 67% and 37% for patients with prognostic scores of 0, 1, 2 and 3, respectively, (no patients with score of 4). Even in the presence of poor disease characteristics, nilotinib provided significant clinical benefit in patients with imatinib-resistant or -intolerant CML. This system may yield insight on the prognosis of patients.

Interleukin-3 protects Bcr-Abl-transformed hematopoietic progenitor cells from apoptosis induced by Bcr-Abl tyrosine kinase inhibitors.

Bcr-Abl tyrosine kinase has been validated as a molecular target for the treatment of chronic myelogenous leukemia (CML). More recently, it has been reported that CML patients could develop resistance to the Bcr-Abl tyrosine kinase inhibitor, imatinib (STI571, Gleevec), pointing to the need for development of additional Bcr-Abl tyrosine kinase inhibitors or other therapeutic strategies. It was also found that a significant proportion of patients who received the Bcr-Abl inhibitor did not achieve complete cytogenetic response. Mechanisms for incomplete cytogenetic response to Bcr-Abl inhibition are not entirely clear. We report here three new pyrido[2,3-d]pyrimidine Bcr-Abl tyrosine kinase inhibitors, PD164199, PD173952, PD173958, that induced apoptosis of Bcr-Abl-dependent hematopoietic cells. An interleukin-3 (IL-3) autocrine loop was observed previously in primitive CD34(+)/Bcr-Abl(+) leukemic cells in CML patients. Using 32Dp210(Bcr-Abl)and Baf3p210(Bcr-Abl) cells as models, we tested whether IL-3 might protect Bcr-Abltransformed, IL-3-responsive cells from apoptosis caused by Bcr-Abl tyrosine kinase inhibition. Results of trypan blue exclusion, fluoroisothiocyanate-valyl-alanyl-aspartyl-[O-methyl] -fluoromethylketone (FITC-VAD-FMK), and Annexin-V/7-amino-actinomycin D (7-AAD) binding assays indicate that IL-3 could protect Bcr-Abl-transformed, IL-3 responsive hematopoietic progenitor cells from apoptosis induced by Bcr-Abl tyrosine kinase inhibitors. This finding raises the possibility that the IL-3 autocrine loop found in primitive CD34(+)/Bcr-Abl(+) cells in CML patients could contribute to the incomplete eradication of Bcr-Abl(+) cells by Bcr-Abl inhibition.

The Hsp90 inhibitor geldanamycin selectively sensitizes Bcr-Abl-expressing leukemia cells to cytotoxic chemotherapy.

The Bcr-Abl fusion protein drives leukemogenesis and can render leukemia cells resistant to conventional chemotherapy. Geldanamycin (GA), a drug which destabilizes Hsp90-associated proteins, depletes cells of Bcr-Abl, an Hsp90 client, but not of Abl. Both HL60 cells transfected with Bcr-Abl and naturally Ph1-positive K562 leukemia cells are resistant to most cytotoxic drugs, but were found to be sensitive to GA. Furthermore, GA sensitized Bcr-Abl-expressing cells to doxorubicin (DOX) and paclitaxel (PTX). In contrast, in parental HL60 cells, 90 nM GA inhibited PARP cleavage, nuclear fragmentation, and cell death caused by 500 ng/ml DOX. Like GA, STI 571 (an inhibitor of the Abl kinase) sensitized Bcr-Abl-expressing cells to DOX. Unlike GA, STI 571 did not antagonize the cytotoxic effects of DOX in parental HL60 cells. These results indicate that sensitization of Bcr-Abl-expressing cells, but not desensitization of HL60 cells, depends on inhibition of Bcr-Abl. Thus, GA differentially affects leukemia cells depending on their Bcr-Abl expression and selectively increases apoptosis in Bcr-Abl-expressing cells.

Co-treatment with As2O3 enhances selective cytotoxic effects of STI-571 against Brc-Abl-positive acute leukemia cells.

By inhibiting the tyrosine kinase (TK) activity of Bcr-Abl, STI-571 induces differentiation and apoptosis of HL-60/Bcr-Abl (with ectopic expression of p185 Bcr-Abl) and K562 (containing endogenous expression of p210 Bcr-Abl) but not of the control HL-60 cells. Treatment with arsenic trioxide (As2O3) lowers Bcr-Abl protein levels and induces apoptosis of the Bcr-Abl-positive leukemic blasts (Blood 2000; 95: 1014). Here, we demonstrate that compared to treatment with STI-571 (0.25 to 1.0 microM) or As2O3 (0.5 to 2.0 microM) alone, combined treatment with As2O3 and STI-571 induced significantly more apoptosis of HL-60/Bcr-Abl and K562 but not HL-60/neo cells (P < 0.05). Combined treatment with As2O3 and STI-571 also resulted in greater reductions in the levels of Bcl-x(L), XIAP and Akt, and inhibition of Akt kinase activity. Co-treatment with As2O3 inhibited STI-571-induced hemoglobin, which was associated with the cleavage and downregulation of GATA-1 transcription factor involved in erythroid differentiation. These data demonstrate that a treatment strategy which combines an agent that lowers Bcr-Abl levels, eg As2O3, with an agent that inhibits Bcr-Abl TK activity, eg STI-571, can potently induce apoptosis and differentiation of Bcr-Abl-positive human leukemic cells.

Pretreatment with paclitaxel enhances apo-2 ligand/tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of prostate cancer cells by inducing death receptors 4 and 5 protein levels.

We have demonstrated that Apo-2 ligand (Apo-2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis of human prostate cancer PC-3, DU145, and LNCaP cells in a dose-dependent manner, with PC-3 cells displaying the greatest sensitivity to Apo-2L/TRAIL. Susceptibility of the prostate cancer cell types to Apo-2L/TRAIL-induced apoptosis did not appear to correlate with the levels of the Apo-2L/TRAIL receptors death receptor (DR) 4 (TRAIL receptor 1) or DR5 (TRAIL receptor 2), decoy receptor (DcR) 1 and DcR2, Flame-1, or the inhibitors of apoptosis proteins family of proteins. Apo-2L/TRAIL-induced apoptosis of PC-3 cells was associated with the processing of caspase-8, caspase-10, and the proapoptotic Bid protein, resulting in the cytosolic accumulation of cytochrome c as well as the processing of procaspase-9 and procaspase-3. Cotreatment with the caspase-8 inhibitor z-IETD-fmk or DR4:Fc significantly inhibited Apo-2L/TRAIL-induced apoptosis. Treatment with paclitaxel or taxotere increased DR4 and/or DR5 protein levels (up to 8-fold) without affecting the protein levels of DcR1 and DcR2, Apo-2L/TRAIL, Fas, or Fas ligand. Up-regulation of DR4 and DR5 was not preceded by the induction of their mRNA levels but was inhibited by cotreatment with cycloheximide. Importantly, sequential treatment of PC-3, DU145, and LNCaP cells with paclitaxel followed by Apo-2L/TRAIL induced significantly more apoptosis than Apo-2L/TRAIL treatment alone (P < 0.01). This was also associated with greater processing of procaspase-8 and Bid, as well as greater cytosolic accumulation of cytochrome c and the processing of caspase-3. These findings indicate that up-regulation of DR4 and DR5 protein levels by treatment with paclitaxel enhances subsequent Apo-2L/TRAIL-induced apoptosis of human prostate cancer cells.

CGP57148B (STI-571) induces differentiation and apoptosis and sensitizes Bcr-Abl-positive human leukemia cells to apoptosis due to antileukemic drugs.

The differentiation and apoptosis-sensitizing effects of the Bcr-Abl-specific tyrosine kinase inhibitor CGP57148B, also known as STI-571, were determined in human Bcr-Abl-positive HL-60/Bcr-Abl and K562 cells. First, the results demonstrate that the ectopic expression of the p185 Bcr-Abl fusion protein induced hemoglobin in the acute myeloid leukemia (AML) HL-60 cells. Exposure to low-dose cytosine arabinoside (Ara-C; 10 nmol/L) increased hemoglobin levels in HL-60/Bcr-Abl and in the chronic myeloid leukemia (CML) blast crisis K562 cells, which express the p210 Bcr-Abl protein. As compared with HL-60/neo, HL-60/Bcr-Abl and K562 cells were resistant to apoptosis induced by Ara-C, doxorubicin, or tumor necrosis factor-alpha (TNF-alpha), which was associated with reduced processing of caspase-8 and Bid protein and decreased cytosolic accumulation of cytochrome c (cyt c). Exposure to CGP57148B alone increased hemoglobin levels and CD11b expression and induced apoptosis of HL-60/Bcr-Abl and K562 cells. CGP57148B treatment down-regulated antiapoptotic XIAP, cIAP1, and Bcl-x(L), without affecting Bcl-2, Bax, Apaf-1, Fas (CD95), Fas ligand, Abl, and Bcr-Abl levels. CGP57148B also inhibited constitutively active Akt kinase and NFkappaB in Bcr-Abl-positive cells. Attenuation of NFkappaB activity by ectopic expression of transdominant repressor of IkappaB sensitized HL-60/Bcr-Abl and K562 cells to TNF-alpha but not to apoptosis induced by Ara-C or doxorubicin. Importantly, cotreatment with CGP57148B significantly increased Ara-C- or doxorubicin-induced apoptosis of HL-60/Bcr-Abl and K562 cells. This was associated with greater cytosolic accumulation of cyt c and PARP cleavage activity of caspase-3. These in vitro data indicate that combinations of CGP57148B and antileukemic drugs such as Ara-C may have improved in vivo efficacy against Bcr-Abl-positive acute leukemia.

The role of Apaf-1, caspase-9, and bid proteins in etoposide- or paclitaxel-induced mitochondrial events during apoptosis.

Ectopic overexpression of Apaf-1 (2.5-fold) in human acute myelogenous leukemia HL-60 cells (HL-60/Apaf-1 cells) induced apoptosis and sensitized HL-60/Apaf-1 cells to etoposide- and paclitaxel-induced apoptosis (C. Perkins et al., Cancer Res., 58: 4561-4566, 1998). In this report, we demonstrate that in HL-60/Apaf-1 cells, the activity of caspase-9 and -3 induced by Apaf-1 overexpression was associated with a significant increase (5-fold) in the cytosolic accumulation of cytochrome c (cyt c), loss of mitochondrial membrane potential (deltapsim), and an increase in the reactive oxygen species. These were also associated with the processing of procaspase-8 and Bid (cytosolic, proapoptotic BH3 domain containing protein). Transient transfection of Apaf-1 into the Apaf-1-containing mouse embryogenic fibroblasts (MEFs; Apaf-1+/- MEFs) or Apaf-1-/- MEFs also induced the processing of procaspase-9 and procaspase-8, Bid cleavage, and apoptosis. These events were secondary to the activity of the downstream caspases induced by Apaf-1. This conclusion is supported by the observation that in HL-60/Apaf-1 cells, ectopic expression of dominant negative caspase-9, its inhibitory short isoform caspase-9b, or XIAP or treatment with the caspase inhibitor zVAD (50 microM) inhibited Apaf-1-induced caspase-8 and Bid cleavage, mitochondrial deltapsim, release of cyt c, and apoptosis. In contrast, a transient transfection of dominant negative caspase-8 or CrmA or exposure to caspase-8 inhibitor zIETD-fmk inhibited the processing of procaspase-8 and Bid but did not inhibit the cytosolic accumulation of cyt c in either the untreated HL-60/Apaf-1 cells or the etoposide-treated HL-60/Apaf-1 and HL-60/neo cells. These results indicate that Apaf-1 overexpression lowers the apoptotic threshold by activating caspase-9 and caspase-3. This triggers the mitochondrial deltapsim and cyt c release into the cytosol through a predominant mechanism other than cleavage of caspase-8 and/or Bid. This mechanism may involve a cytosolic mitochondrial permeability transition factor, which may be processed and activated by the downstream effector caspases, thereby completing an amplifying feedback loop, which triggers the mitochondrial events during apoptosis.

Arsenic induces apoptosis of multidrug-resistant human myeloid leukemia cells that express Bcr-Abl or overexpress MDR, MRP, Bcl-2, or Bcl-x(L).

We investigated the in vitro growth inhibitory and apoptotic effects of clinically achievable concentrations of As(2)O(3) (0.5 to 2.0 micromol/L) against human myeloid leukemia cells known to be resistant to a number of apoptotic stimuli. These included chronic myelocytic leukemia (CML) blast crisis K562 and HL-60/Bcr-Abl cells, which contain p210 and p185 Bcr-Abl, respectively, and HL-60 cell types that overexpress Bcl-2 (HL-60/Bcl-2), Bcl-x(L) (HL-60/Bcl-x(L)), MDR (HL-60/VCR), or MRP (HL-60/AR) protein. The growth-inhibitory IC(50) values for As(2)O(3) treatment for 7 days against all these cell types ranged from 0.8 to 1.5 micromol/L. Exposure to 2 micromol/L As(2)O(3) for 7 days induced apoptosis of all cell types, including HL-60/Bcr-Abl and K562 cells. This was associated with the cytosolic accumulation of cyt c and preapoptotic mitochondrial events, such as the loss of inner membrane potential (DeltaPsim) and the increase in reactive oxygen species (ROS). Treatment with As(2)O(3) (2 micromol/L) generated the activities of caspases, which produced the cleavage of the BH3 domain containing proapoptotic Bid protein and poly (ADP-ribose) polymerase. Significantly, As(2)O(3)-induced apoptosis of HL-60/Bcr-Abl and K562 cells was associated with a decline in Bcr-Abl protein levels, without any significant alterations in the levels of Bcl-x(L), Bax, Apaf-1, Fas, and FasL. Although As(2)O(3 )treatment caused a marked increase in the expression of the myeloid differentiation marker CD11b, it did not affect Hb levels in HL-60/Bcr-Abl, K562, or HL-60/neo cells. However, in these cells, As(2)O(3 )potently induced hyper-acetylation of the histones H3 and H4. These findings characterize As(2)O(3) as a growth inhibiting and apoptosis-inducing agent against a variety of myeloid leukemia cells resistant to multiple apoptotic stimuli.

Phase I and pharmacologic study of a 3-hour infusion of paclitaxel followed by cisplatinum and 5-fluorouracil in patients with advanced solid tumors.

A Phase I and pharmacological study of paclitaxel administered as an outpatient, 3-h i.v. infusion just before a 5-day regimen of daily cisplatinum (CP) and a continuous infusion of 5-fluorouracil (5-FU) was performed in patients with advanced solid tumors. A secondary objective was to determine the objective response rate to this regimen. Forty-two patients were enrolled and were evaluable for toxicities. Eighteen patients were previously untreated, whereas the rest had received prior treatment with radiation (J. H. Schiller et al., J. Clin. Oncol., 12: 241-248, 1994), chemotherapy (M. J. Kennedy et al., Clin. Cancer Res., 4: 349-356, 1998), or both modalities (J. H. Schiller et al., J. Clin. Oncol., 12: 241-248, 1994). The paclitaxel dose was escalated from 100-135-170-200-225 to 250 mg/m2, whereas i.v. 5-FU and CP doses were fixed at 1.0 g/m2/day continuous infusion and 20 mg/m2/day, respectively, daily for 5 days. Granulocyte colony-stimulating factor (G-CSF; 5 microg/kg/day) was administered s.c. from day 6, routinely after 250 mg/m2 dose of paclitaxel or after a lower dose of paclitaxel if ANC <500/microl or febrile neutropenia was observed. Patients were treated every 28 days. Plasma and urine samples were collected to determine the pharmacokinetics of paclitaxel. In previously untreated patients, the maximally tolerated dose of paclitaxel in the drug regimen was determined to be 170 mg/m2 without and 250 mg/m2 with G-CSF support. At the higher dose level, mucositis and thrombocytopenia were dose-limiting. In previously treated patients, these toxicities were observed at all dose levels of paclitaxel > or =135 mg/m2. With increasing doses of paclitaxel, a disproportionate increase in the peak concentrations, as well as the area under plasma concentration time-curve, was seen. This nonlinearity was due to saturable total body clearance and volume of distribution of paclitaxel (P < 0.001). The apparent plasma elimination half-life was unaffected by the dose of paclitaxel. CP and 5-FU had no apparent effect on the metabolism of paclitaxel. Among 32 patients evaluable for response, 22 demonstrated an objective response, including five complete remissions. Therefore, a regimen of 3-h infusion of 250 mg/m2 paclitaxel before CP and FU is tolerable with G-CSF (as above) support in previously untreated patients. The regimen also seems to be highly active against breast and esophageal cancers.

'Loop' domain deletional mutant of Bcl-xL is as effective as p29Bcl-xL in inhibiting radiation-induced cytosolic accumulation of cytochrome c (cyt c), caspase-3 activity, and apoptosis.

PURPOSE/OBJECTIVE: To investigate the effect of the enforced expression of p29Bcl-xL or its loop deletional mutant, p18Bcl-xLdelta, on irradiation-induced apoptosis and cell-cycle distribution of HL-60 cells. MATERIALS & METHODS: We compared the irradiation-induced molecular cascade of apoptosis in control human AML HL-60/neo versus Bcl-xL overexpressing (approximately 8-fold) (HL-60/Bcl-xL) and HL-60/Bcl-XLdelta cells that express the loop domain deletional mutant construct (delta26-83 AA) of Bcl-xL. The three cell lines were irradiated with 6MV photons to varying doses up to 20 Gy. Following this, cytosolic cyt c levels, caspase-3 activity, and the Bcl-2 family of proteins were evaluated utilizing Western blot analysis (whole cell lysate or cytosolic S-100 fraction). Apoptosis was assessed by internucleosomal DNA fragmentation, Annexin-V staining and FACS analysis, as well as by morphologic criteria. The cell-cycle effects of radiation were analyzed by flow cytometry. RESULTS: Eight hours following irradiation (12 Gy) of HL-60/neo cells, a marked increase (approximately 8-fold) in the cytosolic accumulation of cyt c in the S-100 fraction was observed. This was associated with the cleavage of caspase-3, as well as the generation of its poly (ADP-ribose) polymerase (PARP) and DFF (DNA fragmentation factor)-45 cleavage activity. Twenty-four to forty-eight hours after irradiation, internucleosomal DNA fragmentation and positive Annexin-V staining (32.3+/-3.3%) was detected in HL-60/neo cells. In contrast, in both HL-60/Bcl-xL and HL-60/Bcl-xLdelta cells, a significantly lower percentage of apoptotic cells (p<0.05) were detected and internucleosomal DNA fragmentation was not induced. Following irradiation, Western analysis neither demonstrated any significant alteration in Bcl-2, p29Bcl-xL, p18Bcl-xLdelta, or Bax; nor induced CD95 (Fas receptor) or Fas ligand expression in any cell type. However, in all cell types, irradiation produced approximately a 2-fold increase in the percentage of cells in the G2/M phase of the cell cycle. CONCLUSION: These results demonstrate that an intact loop domain is not necessary for the full antiapoptotic function of Bcl-xL against irradiation-induced cytosolic accumulation of cyt c, caspase activation, and apoptosis of HL-60 cells. Additionally, the cell-cycle effects of ionizing radiation in HL-60 cells are not affected by enforced expression of Bcl-xL or Bcl-xLdelta.

Overexpression of Apaf-1 promotes apoptosis of untreated and paclitaxel- or etoposide-treated HL-60 cells.

Recent studies have demonstrated that Apaf-1 is the adaptor molecule which in the presence of cytosolic cytochrome c (cyt c) and dATP interacts with procaspase-9, resulting in the sequential cleavage and activity of caspase-9 and caspase-3, followed by apoptosis. In the present studies, we determined the effect of enforced overexpression of Apaf-1 on the apoptotic threshold in the human myeloid leukemia HL-60 cells. Our findings demonstrate that both transient and stable transfections resulted in a 2.5-fold higher expression of Apaf-1, which was associated with approximately a 5-fold increase in the percentage of apoptosis in the transfectants (HL-60/Apaf-1) as compared with the control HL-60/neo cells. In cells overexpressing either Bcl-2 or Bcl-xL, transient overexpression of Apaf-1 did not induce apoptosis. Stably overexpressing Apaf-1 levels significantly sensitized HL-60/Apaf-1 cells to apoptosis induced by clinically achievable concentrations of paclitaxel or etoposide (P < 0.01). This increase in paclitaxel- or etoposide-induced apoptosis of HL-60/Apaf-1 cells was not associated with any significant alterations in Bcl-2, Bcl-xL, Bax, Fas, or Fas ligand expression. It was, however, clearly associated with caspase-9 cleavage, as well as the poly(ADP-ribose) polymerase and DFF45 cleavage activity of caspase-3. Coexpression of the catalytically inactive, dominant-negative, mutant caspase-9, XIAP, or treatment with the caspase inhibitor, zVAD, significantly inhibited the increase in apoptosis of HL-60/Apaf-1 cells (P < 0.01). These data indicate that the intracellular levels of Apaf-1 is an important molecular determinant of the threshold for apoptosis induced by paclitaxel and etoposide.

"Loop" domain is necessary for taxol-induced mobility shift and phosphorylation of Bcl-2 as well as for inhibiting taxol-induced cytosolic accumulation of cytochrome c and apoptosis.

Taxol, 1-beta-D-arabinofuranosylcytosine (ara-C), and etoposide induce apoptosis in HL-60 cells that is blocked by overexpression of Bcl-2 or Bcl-xL.A 60-amino acid "loop" domain of Bcl-2 and Bcl-xL that contains phosphorylation sites is known to negatively regulate their antiapoptotic function. In the present studies, Taxol-, ara-C-, or etoposide-induced apoptosis was examined in HL-60/Bcl-2delta and HL-60/Bcl-xLdelta cells that express the loop-deletional mutant cDNA constructs p19Bcl-2delta32-80 and p18Bcl-xLdelta26-83, respectively. This was compared with control HL-60/neo cells as well as HL-60/Bcl-2 and HL-60/Bcl-xL cells. The latter two cell lines overexpress full-length Bcl-2 and Bcl-xL, respectively. Immunoblot analyses showed that HL-60/neo and HL-60/Bcl-2delta cells express similar levels of p26Bcl-2. In contrast, as compared with HL-60/neo, HL-60/Bcl-xLdelta cells expressed significantly lower levels of p26Bcl-2. p29Bcl-xL and p21Bax levels were similar in all cell types. Exposure to etoposide (50 microM) or ara-C (100 microM) for 4 h induced apoptosis in HL-60/neo cells, but not in HL-60/Bcl-2, HL-60/Bcl-xL, HL-60/Bcl-2delta, or HL-60/Bcl-xLdelta cells. In contrast, Taxol treatment (500 nM for 24 h) triggered the molecular cascade of apoptosis, represented by the cytosolic increase of cytochrome c and poly(ADP-ribose) polymerase or the DNA fragmentation factor cleavage activity of caspase-3 in HL-60/neo cells as well as in HL-60/Bcl-xLdelta and HL-60/Bcl-2delta cells, but not in their counterparts overexpressing full-length Bcl-2 and Bcl-xL. Equal amounts of p26Bcl-2 were coimmunoprecipitated with apoptosis protease-activating factor 1 (APAF-1) in HL-60/neo and HL-60/Bcl-2delta cells, whereas a markedly higher level of p26Bcl-2 coimmunoprecipitated with APAF-1 in HL-60/Bcl-2 cells. In association with Taxol-induced apoptosis, the levels of Bcl-2 that were coimmunoprecipitated with APAF-1 declined in HL-60/neo and HL-60/Bcl-2delta cells. This was not observed in HL-60/Bcl-2 cells, in which Taxol-induced apoptosis was blocked. Previous studies have demonstrated that Taxol induces phosphorylation of Bcl-2 in association with Taxol-induced apoptosis of HL-60/neo cells. Immunoblot analysis demonstrated a Taxol-induced mobility shift of Bcl-2 but not p19Bcl-2delta. Taxol also increased [32P]Pi incorporation in p26Bcl-2, but not in p19Bcl-2delta or p18Bcl-xL. These findings indicate that the loop domain is necessary for the Taxol-induced mobility shift and phosphorylation of Bcl-2. Loop domain also seems to be necessary for the antiapoptotic effect of Bcl-2 against Taxol-induced apoptosis but not ara-C- or etoposide-induced apoptosis.