pyrimidinones and Leukemia--Myeloid--Acute

Belinostat is an intravenous histone deacetylase inhibitor with approval for T-cell lymphomas. Adavosertib is a first in class oral Wee1 inhibitor. Preclinical studies of the combination demonstrated synergy in various human acute myeloid leukemia (AML) lines as well as AML xenograft mouse models.. This was a phase 1 dose-escalation study of belinostat and adavosertib in patients with relapsed/refractory AML and myelodysplastic syndrome (MDS). Patients received both drugs on days 1-5 and 8-12 of a 21-day cycle. Safety and toxicity were monitored throughout the study. Plasma levels of both drugs were measured for pharmacokinetic analysis. Response was determined by standard criteria including bone marrow biopsy.. The combination of belinostat and adavosertib at the tested dose levels was feasible but without efficacy signals in the relapsed/refractory MDS/AML population.

Topics: Animals; Humans; Hydroxamic Acids; Leukemia, Myeloid, Acute; Mice; Pyrimidinones

RAS pathway mutations are common mechanisms of resistance to acute myeloid leukemia (AML) therapies. Trametinib, an oral MEK inhibitor, has been shown to have single-agent activity in relapsed/refractory AML and preclinical synergy with venetoclax.. We conducted a single-center, open-label, phase 2 trial of the combination of azacitidine, venetoclax, and trametinib in patients with relapsed or refractory AML harboring a RAS pathway-activating mutation.. Sixteen patients were treated. The patients were heavily pretreated with a median number of 4 prior therapies; 13 (81%) had received a prior hypomethylating agent (HMA) with venetoclax, and 8 (50%) had undergone prior stem cell transplant. Four patients (25%) responded (CR, n = 1; CRi, n = 1; MLFS, n = 2). Two of the 3 patients (67%) who had not previously received HMA plus venetoclax responded; in contrast, only 2 of the 13 patients (15%) who had previously received HMA plus venetoclax responded. The median OS was 2.4 months, and the 6-month OS rate was 31%. Related grade 3-4 adverse events occurred in 50% of patients, and 50% of patients required a dose adjustment of trametinib.. The combination of azacitidine, venetoclax, and trametinib had only modest activity in patients with relapsed/refractory AML, with a response rate that was similar to previous reports of trametinib monotherapy. Substantial toxicity was observed with this combination. Given the established role of RAS pathway mutations in mediating resistance to AML therapies, future studies of better tolerated, more active inhibitors of this pathway are still needed.

Topics: Antineoplastic Combined Chemotherapy Protocols; Azacitidine; Bridged Bicyclo Compounds, Heterocyclic; Humans; Leukemia, Myeloid, Acute; Mitogen-Activated Protein Kinase Kinases; Mutation; Pyridones; Pyrimidinones; Sulfonamides

FLT3 mutations are prevalent in AML patients and confer poor prognosis. Crenolanib, a potent type I pan-FLT3 inhibitor, is effective against both internal tandem duplications and resistance-conferring tyrosine kinase domain mutations. While crenolanib monotherapy has demonstrated clinical benefit in heavily pretreated relapsed/refractory AML patients, responses are transient and relapse eventually occurs. Here, to investigate the mechanisms of crenolanib resistance, we perform whole exome sequencing of AML patient samples before and after crenolanib treatment. Unlike other FLT3 inhibitors, crenolanib does not induce FLT3 secondary mutations, and mutations of the FLT3 gatekeeper residue are infrequent. Instead, mutations of NRAS and IDH2 arise, mostly as FLT3-independent subclones, while TET2 and IDH1 predominantly co-occur with FLT3-mutant clones and are enriched in crenolanib poor-responders. The remaining patients exhibit post-crenolanib expansion of mutations associated with epigenetic regulators, transcription factors, and cohesion factors, suggesting diverse genetic/epigenetic mechanisms of crenolanib resistance. Drug combinations in experimental models restore crenolanib sensitivity.

Topics: Adult; Aged; Aged, 80 and over; Animals; Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Epigenesis, Genetic; Exome Sequencing; Female; fms-Like Tyrosine Kinase 3; GTP Phosphohydrolases; HEK293 Cells; Humans; Inhibitory Concentration 50; Isocitrate Dehydrogenase; Leukemia, Myeloid, Acute; Male; Membrane Proteins; Mice; Middle Aged; Mutation; Piperidines; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; Tandem Repeat Sequences; Treatment Outcome

With proven single-agent activity and favorable toxicity profile of MEK-1/2 inhibition in advanced leukemia, investigation into combination strategies to overcome proposed resistance pathways is warranted. Resistance to MEK inhibition is secondary to upstream hyperactivation of RAS/RAF or activation of the PI3K/PTEN/AKT/mTOR pathway. This phase II multi-institution Cancer Therapy Evaluation Program-sponsored study was conducted to determine efficacy and safety of the combination of the ATP-competitive pan-AKT inhibitor GSK2141795, targeting the PI3K/AKT pathway, and the MEK inhibitor trametinib in RAS-mutated relapsed/refractory acute myeloid leukemia (AML).. The primary objective was to determine the proportion of patients achieving a complete remission. Secondary objectives included assessment of toxicity profile and biologic effects of this combination. Twenty-three patients with RAS-mutated AML received the combination. Two dose levels were explored (dose level 1: 2 mg trametinib, 25 mg GSK2141795 and dose level 2: 1.5 mg trametinib, 50 mg GSK2141795).. Dose level 1 was identified as the recommended phase II dose. No complete remissions were identified in either cohort. Minor responses were recognized in 5 (22%) patients. The most common drug-related toxicities included rash and diarrhea, with dose-limiting toxicities of mucositis and colitis. Longitudinal correlative assessment of the modulation of MEK and AKT pathways using reverse phase protein array and phospho-flow analysis revealed significant and near significant down-modulation of pERK and pS6, respectively. Combined MEK and AKT inhibition had no clinical activity in patients with RAS-mutated AML.. Further investigation is required to explore the discrepancy between the activity of this combination on leukemia cells and the lack of clinical efficacy.

Topics: Administration, Oral; Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Diamines; Female; Genes, ras; Humans; Leukemia, Myeloid, Acute; Male; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Middle Aged; Mutation; Proto-Oncogene Proteins c-akt; Pyrazoles; Pyridones; Pyrimidinones; Treatment Outcome; Young Adult

Topics: Acetates; Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Molecular Targeted Therapy; Piperidones; Proto-Oncogene Proteins c-mdm2; Pyridones; Pyrimidinones; Recurrence; Treatment Outcome

RAS/RAF/mitogen-activated protein kinase activation is common in myeloid malignancies. Trametinib, a mitogen-activated protein kinase kinase 1 (MEK1)/MEK2 inhibitor with activity against multiple myeloid cell lines at low nanomolar concentrations, was evaluated for safety and clinical activity in patients with relapsed/refractory leukemias.. This phase 1/2 study accrued patients with any relapsed/refractory leukemia in phase 1. In phase 2, this study accrued patients with relapsed/refractory acute myeloid leukemia (AML) or high-risk myelodysplastic syndromes (MDS) with NRAS or KRAS mutations (cohort 1); patients with AML, MDS, or chronic myelomonocytic leukemia (CMML) with a RAS wild-type mutation or an unknown mutation status (cohort 2); and patients with CMML with an NRAS or KRAS mutation (cohorts 3).. The most commonly reported treatment-related adverse events were diarrhea, rash, nausea, and increased alanine aminotransferase levels. The phase 2 recommended dose for Trametinib was 2 mg orally daily. The overall response rates were 20%, 3%, and 27% for cohorts 1, 2, and 3, respectively, and this indicated preferential activity among RAS-mutated myeloid malignancies. Repeated cycles of trametinib were well tolerated with manageable or reversible toxicities; these results were similar to those of other trametinib studies.. The selective, single-agent activity of trametinib against RAS-mutated myeloid malignancies validates its therapeutic potential. Combination strategies based on a better understanding of the hierarchical role of mutations and signaling in myeloid malignancies are likely to improve the response rate and duration. Cancer 2016;122:1871-9. © 2016 American Cancer Society.

Topics: Adult; Aged; Aged, 80 and over; Dose-Response Relationship, Drug; Humans; Kaplan-Meier Estimate; Leukemia; Leukemia, Myeloid, Acute; MAP Kinase Signaling System; Middle Aged; Mutation; Myelodysplastic Syndromes; Protein Kinase Inhibitors; Proto-Oncogene Proteins p21(ras); Pyridones; Pyrimidinones; Recurrence; Young Adult

Eg5 (kinesin spindle protein) is a microtubule motor protein, essential for centrosome separation during mitosis. This Phase I/II, open-label, multicenter, two-part study investigated AZD4877, a potent Eg5 inhibitor, in patients with acute myeloid leukemia. Primary objectives were to determine the maximum tolerated dose (MTD) (part A), assess efficacy (part B) and determine the pharmacokinetic profile (parts A and B). Secondary objectives included assessment of safety and tolerability. AZD4877 was administered at a range of doses (2, 4, 7, 10, 13, 16 and 18 mg/day) as a 1-hour intravenous infusion on three consecutive days of a continuous 2-week schedule. The MTD in part A was defined as 16 mg/day based on dose-limiting stomatitis at 16 and 18 mg/day, hyperbilirubinemia at 16 mg/day and palmar-plantar erythrodysesthesia syndrome at 18 mg/day. Systemic exposure to AZD4877 generally increased with increasing dose whereas half-life was not dose dependent. No evaluable patients experienced a complete remission (CR) or CR with incomplete blood count recovery (CRi), demonstrating no evidence of AZD4877 efficacy in this population. Evidence of monoasters in all but the 4 mg/day dose group provided proof of mechanism for AZD4877. This study was terminated due to lack of efficacy. (ClinicalTrials.gov identifier NCT00486265).

Topics: Adult; Aged; Aged, 80 and over; Antimitotic Agents; Benzamides; Female; Humans; Kinesins; Leukemia, Myeloid, Acute; Male; Middle Aged; Pyrimidinones; Young Adult

AMP-activated protein kinase (AMPK) regulates the balance between cellular anabolism and catabolism dependent on energy resources to maintain proliferation and survival. Small-compound AMPK activators show anti-cancer activity in preclinical models. Using the direct AMPK activator GSK621, we show that the unfolded protein response (UPR) is activated by AMPK in acute myeloid leukemia (AML) cells. Mechanistically, the UPR effector protein kinase RNA-like ER kinase (PERK) represses oxidative phosphorylation, tricarboxylic acid (TCA) cycle, and pyrimidine biosynthesis and primes the mitochondrial membrane to apoptotic signals in an AMPK-dependent manner. Accordingly, in vitro and in vivo studies reveal synergy between the direct AMPK activator GSK621 and the Bcl-2 inhibitor venetoclax. Thus, selective AMPK-activating compounds kill AML cells by rewiring mitochondrial metabolism that primes mitochondria to apoptosis by BH3 mimetics, holding therapeutic promise in AML.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; AMP-Activated Protein Kinases; Animals; Antineoplastic Agents; Apoptosis; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Citric Acid Cycle; Drug Evaluation, Preclinical; eIF-2 Kinase; Female; HEK293 Cells; HL-60 Cells; Humans; Imidazoles; Leukemia, Myeloid, Acute; Male; Mice; Middle Aged; Mitochondria; Oxidative Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Pyrimidinones; Sulfonamides; THP-1 Cells; U937 Cells; Unfolded Protein Response; Young Adult

Topics: Antineoplastic Combined Chemotherapy Protocols; Bridged Bicyclo Compounds, Heterocyclic; Humans; Leukemia, Myeloid, Acute; Neoplasm Recurrence, Local; Pyridones; Pyrimidinones; Sulfonamides

All-trans retinoic acid (ATRA) is only clinically useful in acute promyelocytic leukemia (APL), but not other subtypes of acute myeloid leukemia (AML). In the present study, a clinically achievable concentration of trametinib, a highly selective inhibitor of MEK, enhanced ATRA-induced differentiation in AML cell lines, HL-60 and U937 as well as AML primary cells. Moreover, trametinib-ATRA (tra-ATRA) co-treatment restored ATRA sensitivity in ATRA-resistant AML cell line, HL-60Res. The protein level of STAT3 and the phosphorylation of Akt or JNK were enhanced with tra-ATRA treatment in HL-60, U937, and HL-60Res cells, respectively. Furthermore, tra-ATRA-induced differentiation in HL-60, U937, and HL-60Res cells was inhibited by STAT3, PI3K, and JNK inhibitors, respectively. Therefore, STAT3, Akt, and JNK signaling pathways were involved in tra-ATRA-induced differentiation in HL-60, U937, and HL-60Res cells, respectively. Taken together, our findings may provide novel therapeutic strategies for AML patients.

Topics: Cell Differentiation; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Proto-Oncogene Proteins c-akt; Pyridones; Pyrimidinones; Tretinoin

FMS-like tyrosine kinase 3 (FLT3) is a key driver of acute myeloid leukemia (AML). Several tyrosine kinase inhibitors (TKIs) targeting FLT3 have been evaluated clinically, but their effects are limited when used in monotherapy due to the emergence of drug-resistance. Thus, a better understanding of drug-resistance pathways could be a good strategy to explore and evaluate new combinational therapies for AML. Here, we used phosphoproteomics to identify differentially-phosphorylated proteins in patients with AML and TKI resistance. We then studied resistance mechanisms in vitro and evaluated the efficacy and safety of rational combinational therapy in vitro, ex vivo and in vivo in mice. Proteomic and immunohistochemical studies showed the sustained activation of ERK1/2 in bone marrow samples of patients with AML after developing resistance to FLT3 inhibitors, which was identified as a common resistance pathway. We examined the concomitant inhibition of MEK-ERK1/2 and FLT3 as a strategy to overcome drug-resistance, finding that the MEK inhibitor trametinib remained potent in TKI-resistant cells and exerted strong synergy when combined with the TKI midostaurin in cells with mutated and wild-type FLT3. Importantly, this combination was not toxic to CD34+ cells from healthy donors, but produced survival improvements in vivo when compared with single therapy groups. Thus, our data point to trametinib plus midostaurin as a potentially beneficial therapy in patients with AML.

Topics: Adult; Aged; Animals; Antigens, CD34; Bone Marrow Cells; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Synergism; Female; fms-Like Tyrosine Kinase 3; Humans; Leukemia, Myeloid, Acute; Male; MAP Kinase Kinase Kinase 1; MAP Kinase Signaling System; Mice; Middle Aged; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; Staurosporine

MEK inhibitors are new promising anticancer drugs. The aim of this study was to investigate the effect of the combination treatment of voreloxin with the MEK inhibitor TAK-733 on HL60 myeloid leukemia cells.. MAPK activity, cell viability, apoptosis, oxidative stress induction and AIF (apoptosis-inducing factor) distribution were assessed in HL60 cells cultured with each drug alone or with both drugs.. TAK-733 alone at 5 μM significantly reduced MAPK activity and did not influence viability and apoptosis in HL60 cells. Voreloxin at concentration of 0.03-0.48 μM reduced cell viability and increased apoptosis rate. Incubation with both drugs caused further inhibition of cell viability and increased apoptosis associated with generation of reactive oxygen species (ROS) and nuclear translocation of AIF.. Combination of TAK-733 and voreloxin can exert a synergistic anticancer effect in myeloid leukemia cells.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Inducing Factor; Cell Nucleus; Cell Survival; Drug Synergism; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; MAP Kinase Kinase Kinases; Microscopy, Confocal; Naphthyridines; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; Reactive Oxygen Species; Thiazoles

Targeting of human cancer stem cells (CSCs) requires the identification of vulnerabilities unique to CSCs versus healthy resident stem cells (SCs). Unfortunately, dysregulated pathways that support transformed CSCs, such as Wnt/β-catenin signaling, are also critical regulators of healthy SCs. Using the ICG-001 and CWP family of small molecules, we reveal Sam68 as a previously unappreciated modulator of Wnt/β-catenin signaling within CSCs. Disruption of CBP-β-catenin interaction via ICG-001/CWP induces the formation of a Sam68-CBP complex in CSCs that alters Wnt signaling toward apoptosis and differentiation induction. Our study identifies Sam68 as a regulator of human CSC vulnerability.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Animals; Apoptosis; Azabicyclo Compounds; beta Catenin; Breast Neoplasms; Bridged Bicyclo Compounds, Heterocyclic; Cell Differentiation; Cells, Cultured; Colonic Neoplasms; DNA-Binding Proteins; Female; Humans; Leukemia, Myeloid, Acute; Male; Mice; Mice, Inbred NOD; Middle Aged; Neoplastic Stem Cells; Organophosphates; Peptide Fragments; Proto-Oncogene Proteins c-myc; Pyrimidinones; RNA Interference; RNA-Binding Proteins; Sialoglycoproteins; Sumoylation; Transcriptome; Wnt Signaling Pathway

Although some patients with acute leukemia have good prognoses, the prognosis of adult and pediatric patients who relapse or cannot tolerate standard chemotherapy is poor. Inhibition of WEE1 with AZD1775 has been shown to sensitize cancer cells to genotoxic chemotherapies, including cytarabine in acute myeloid leukemia (AML) and T-ALL. Inhibition of WEE1 impairs homologous recombination by indirectly inhibiting BRCA2. Thus, we sought to determine whether AZD1775 could sensitize cells to the PARP1/2 inhibitor olaparib. We found that combined treatment with AZD1775 and olaparib was synergistic in AML and ALL cells, and this combination impaired proliferative capacity upon drug withdrawal. AZD1775 impaired homologous recombination in olaparib-treated cells, resulting in enhanced DNA damage accumulation and apoptosis induction. This combination enhanced disease control and increased survival in a murine AML model. Furthermore, we demonstrated that combined treatment with AZD1775 and olaparib reduces proliferation and colony formation and increases apoptosis in AML patient samples. In aggregate, these studies raise the possibility of rational combinations of targeted agents for leukemia in patients for whom conventional chemotherapeutics may not be effective or well tolerated.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; DNA Damage; Humans; Leukemia, Myeloid, Acute; Mice; Phthalazines; Piperazines; Pyrazoles; Pyrimidines; Pyrimidinones; Recombinational DNA Repair; Xenograft Model Antitumor Assays

Myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN-U) are rare genetically heterogeneous hematologic diseases associated with older age and a poor prognosis. If the disease progresses into acute myeloid leukemia (AML), it is often refractory to treatment. To gain insight into genetic alterations associated with disease progression, whole exome sequencing and single nucleotide polymorphism arrays were used to characterize the bone marrow and blood samples from a 39-year-old woman at MDS/MPN-U diagnosis and at AML progression, in which routine genetic diagnostics had not identified any genetic alterations. The data revealed the presence of a partial tandem duplication of the MLL gene as the only detectable copy number change and 11 non-silent somatic mutations, including DNMT3A R882H and NRAS G13D. All somatic lesions were present both at initial MDS/MPN-U diagnosis and at AML presentation at similar mutant allele frequencies. The patient has since had two extramedullary relapses and is at high risk of a future bone marrow relapse. A directed ex vivo drug sensitivity analysis showed that the patient's AML cells are sensitive to, for example, the MEK inhibitor trametinib and the proteasome inhibitor bortezomib, indicating that she may benefit from treatment with these drugs. © 2016 Wiley Periodicals, Inc.

Topics: Adult; Bortezomib; Disease Progression; DNA (Cytosine-5-)-Methyltransferases; DNA Methyltransferase 3A; Female; Gene Duplication; Gene Frequency; Genetic Heterogeneity; Genome, Human; GTP Phosphohydrolases; Histone-Lysine N-Methyltransferase; Humans; Leukemia, Myeloid, Acute; Membrane Proteins; Mutation; Myelodysplastic Syndromes; Myeloid-Lymphoid Leukemia Protein; Pyridones; Pyrimidinones

Direct targeting of rat sarcoma (RAS), which is frequently mutated, has proven to be challenging, and inhibition of individual downstream RAS mediators has resulted in limited clinical efficacy. We designed a chemical screen to identify compounds capable of potentiating mammalian target of rapamycin (mTOR) inhibition in mutant RAS-positive leukemia, and identified a Wee1 inhibitor. Synergy was observed in both mutant neuroblastoma RAS viral oncogene homolog (NRAS)- and mutant kirsten RAS viral oncogene homolog (KRAS)-positive acute myelogenous leukemia (AML) cell lines and primary patient samples. The observed synergy enhanced dephosphorylation of AKT, 4E-binding protein 1 and s6 kinase, and correlated with increased apoptosis. The specificity of Wee1 as the target of MK-1775 was validated by Wee1 knockdown, as well as partial reversal of drug combination-induced apoptosis by a cyclin-dependent kinase 1 (CDK1) inhibitor. Importantly, we also extended our findings to other mutant RAS-expressing malignancies, including mutant NRAS-positive melanoma, and mutant KRAS-positive colorectal cancer, pancreatic cancer and lung cancer. We observed favorable responses with combined Wee1/mTOR inhibition in human cancer cell lines from multiple malignancies, and inhibition of tumor growth in in vivo models of mutant KRAS lung cancer and leukemia. The present study introduces for the first time Wee1 inhibition combined with mTOR inhibition as a novel therapeutic strategy for the selective treatment of mutant RAS-positive leukemia and other mutant RAS-expressing malignancies.

Topics: Animals; Antineoplastic Agents; Base Sequence; Cell Cycle Proteins; Cell Line, Tumor; Gene Knockdown Techniques; Leukemia, Myeloid, Acute; Mice; Nuclear Proteins; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras); Pyrazoles; Pyrimidines; Pyrimidinones; RNA, Small Interfering

AMPK is a master regulator of cellular metabolism that exerts either oncogenic or tumor suppressor activity depending on context. Here, we report that the specific AMPK agonist GSK621 selectively kills acute myeloid leukemia (AML) cells but spares normal hematopoietic progenitors. This differential sensitivity results from a unique synthetic lethal interaction involving concurrent activation of AMPK and mTORC1. Strikingly, the lethality of GSK621 in primary AML cells and AML cell lines is abrogated by chemical or genetic ablation of mTORC1 signaling. The same synthetic lethality between AMPK and mTORC1 activation is established in CD34-positive hematopoietic progenitors by constitutive activation of AKT or enhanced in AML cells by deletion of TSC2. Finally, cytotoxicity in AML cells from GSK621 involves the eIF2α/ATF4 signaling pathway that specifically results from mTORC1 activation. AMPK activation may represent a therapeutic opportunity in mTORC1-overactivated cancers.

Topics: AMP-Activated Protein Kinases; Animals; Antineoplastic Agents; Enzyme Activation; Fluorescent Antibody Technique; Heterografts; Humans; Imidazoles; Leukemia, Myeloid, Acute; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Nude; Microscopy, Electron, Transmission; Multiprotein Complexes; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Pyrimidinones; RNA Interference; Signal Transduction; TOR Serine-Threonine Kinases

Topics: AMP-Activated Protein Kinases; Animals; Autophagy; Cell Death; Humans; Imidazoles; Leukemia, Myeloid, Acute; Mechanistic Target of Rapamycin Complex 1; Mice; Molecular Targeted Therapy; Multiprotein Complexes; Pyrimidinones; Sirolimus; TOR Serine-Threonine Kinases

MK-1775 is the first-in-class selective Wee1 inhibitor which has been demonstrated to synergize with CHK1 inhibitors in various malignancies. In this study, we report that the pan-histone deacetylase inhibitor (HDACI) panobinostat synergizes with MK-1775 in acute myeloid leukemia (AML), a malignancy which remains a clinical challenge and requires more effective therapies. Using both AML cell line models and primary patient samples, we demonstrated that panobinostat and MK-1775 synergistically induced proliferation arrest and cell death. We also demonstrated that panobinostat had equal anti-leukemic activities against primary AML blasts derived from patients either at initial diagnosis or at relapse. Interestingly, treatment with panobinostat alone or in combination with MK-1775 resulted in decreased Wee1 protein levels as well as downregulation of the CHK1 pathway. shRNA knockdown of CHK1 significantly sensitized AML cells to MK-1775 treatment, while knockdown of Wee1 significantly enhanced both MK-1775- and panobinostat-induced cell death. Our results demonstrate that panobinostat synergizes with MK-1775 in AML cells, at least in part through downregulation of CHK1 and/or Wee1, providing compelling evidence for the clinical development of the combination treatment in AML.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Checkpoint Kinase 1; Drug Synergism; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Indoles; Inhibitory Concentration 50; Leukemia, Myeloid, Acute; Nuclear Proteins; Panobinostat; Protein Kinase Inhibitors; Protein Kinases; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones

Novel combinations targeting new molecular vulnerabilities are needed to improve the outcome of patients with acute myeloid leukemia. We recently identified WEE1 kinase as a novel target in leukemias. To identify genes that are synthetically lethal with WEE1 inhibition, we performed a short interfering RNA screen directed against cell cycle and DNA repair genes during concurrent treatment with the WEE1 inhibitor MK1775. CHK1 and ATR, genes encoding two replication checkpoint kinases, were among the genes whose silencing enhanced the effects of WEE1 inhibition most, whereas CDK2 short interfering RNA antagonized MK1775 effects. Building on this observation, we examined the impact of combining MK1775 with selective small molecule inhibitors of CHK1, ATR and cyclin-dependent kinases. The CHK1 inhibitor MK8776 sensitized acute myeloid leukemia cell lines and primary leukemia specimens to MK1775 ex vivo, whereas smaller effects were observed with the MK1775/MK8776 combination in normal myeloid progenitors. The ATR inhibitor VE-821 likewise enhanced the antiproliferative effects of MK1775, whereas the cyclin-dependent kinase inhibitor roscovitine antagonized MK1775. Further studies showed that MK8776 enhanced MK1775-mediated activation of the ATR/CHK1 pathway in acute leukemia cell lines and ex vivo. These results indicate that combined cell cycle checkpoint interference with MK1775/MK8776 warrants further investigation as a potential treatment for acute myeloid leukemia.

Topics: Apoptosis; Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; Cell Line, Tumor; Checkpoint Kinase 1; Dose-Response Relationship, Drug; Drug Synergism; Gene Expression Profiling; Gene Silencing; Humans; Leukemia, Myeloid, Acute; Nuclear Proteins; Protein Kinase Inhibitors; Protein Kinases; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones; RNA Interference; RNA, Small Interfering; Signal Transduction; Tumor Stem Cell Assay

Most Down syndrome children with acute myeloid leukemia (DS-AML) have an overall excellent prognosis, however, patients who suffer an induction failure or relapse, have an extremely poor prognosis. Hence, new therapies need to be developed for this subgroup of DS-AML patients. One new therapeutic approach is preventing cell cycle checkpoint activation by inhibiting the upstream kinase wee1 with the first-in-class inhibitor MK-1775 in combination with the standard genotoxic agent cytarabine (AraC).. Using the clinically relevant DS-AML cell lines CMK and CMY, as well as ex vivo primary DS-AML patient samples, the ability of MK-1775 to enhance the cytotoxicity of AraC was investigated with MTT assays. The mechanism by which MK-1775 enhanced AraC cytotoxicity was investigated in the cell lines using Western blots to probe CDK1 and H2AX phosphorylation and flow cytometry to determine apoptosis, cell cycle arrest, DNA damage, and aberrant mitotic entry.. MK-1775 alone had modest single-agent activity, however, MK-1775 was able to synergize with AraC in causing proliferation arrest in both cell lines and primary patient samples, and enhance AraC-induced apoptosis. MK-1775 was able to decrease inhibitory CDK1(Y15) phosphorylation at the relatively low concentration of 100 nM after only 4 hours. Furthermore, it was able to enhance DNA damage induced by AraC and partially abrogate cell cycle arrest. Importantly, the DNA damage enhancement appeared in early S-phase.. MK-1775 is able to enhance the cytotoxicity of AraC in DS-AML cells and presents a promising new treatment approach for DS-AML.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Child; Cytarabine; Down Syndrome; Drug Synergism; Flow Cytometry; Humans; Leukemia, Myeloid, Acute; Nuclear Proteins; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones; Reverse Transcriptase Polymerase Chain Reaction

Acute myeloid leukemia (AML) remains a difficult disease to treat and requires new therapies to improve treatment outcome. Wee1 inhibitors have been used to prevent activation of the G2 cell cycle checkpoint, thus enhancing the antitumor activity of DNA damaging agents. In this study, we investigated MK-1775 in AML cell lines and diagnostic blast samples to identify sensitive subtypes as well as possible mechanisms of resistance.. In vitro MK-1775 cytotoxicities of AML cell lines and diagnostic blasts were measured using MTT assays. The effects of MK-1775 on cell cycle progression and related proteins were determined by propidium iodide (PI) staining and flow cytometry analysis and Western blotting. Drug-induced apoptosis was determined using annexin V/PI staining and flow cytometry analysis.. We found that newly diagnosed and relapsed patient samples were equally sensitive to MK-1775. In addition, patient samples harboring t(15;17) translocation were significantly more sensitive to MK-1775 than non-t(15;17) samples. MK-1775 induced apoptosis in both AML cell lines and diagnostic blast samples, accompanied by decreased phosphorylation of CDK1 and CDK2 on Tyr-15 and increased DNA double-strand breaks (DSBs). Time-course experiments, using AML cell lines, revealed a time-dependent increase in DNA DSBs, activation of CHK1 and subsequent apoptosis following MK-1775 treatment, which could be attenuated by a CDK1/2 inhibitor, Roscovitine. Simultaneous inhibition of CHK1 and Wee1 resulted in synergistic anti-leukemic activity in both AML cell lines and primary patient samples ex vivo.. Our study provides compelling evidence that CHK1 plays a critical role in the anti-leukemic activity of MK-1775 and highlights a possible mechanism of resistance to MK-1775. In addition, our study strongly supports the use of MK-1775 to treat both newly diagnosed and relapsed AML, especially cases with t(15;17) translocation, and supports the development of combination therapies with CHK1 inhibitors.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Checkpoint Kinase 1; Flow Cytometry; Humans; Leukemia, Myeloid, Acute; Nuclear Proteins; Protein Kinases; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones; Tumor Cells, Cultured

Inhibition of Wee1 is emerging as a novel therapeutic strategy for cancer, and some data suggest that cells with dysfunctional p53 are more sensitive to Wee1 inhibition combined with conventional chemotherapy than those with functional p53. We and others found that Wee1 inhibition sensitizes leukemia cells to cytarabine. Thus, we sought to determine whether chemosensitization by Wee1 inhibition is dependent on p53 dysfunction and whether combining Wee1 inhibition is tolerable and effective in vivo. Synergistic inhibition of proliferation with a Wee1 inhibitor in clinical development, MK1775, and cytarabine was observed in all acute myelogenous leukemia (AML) cell lines tested, regardless of p53 functionality. Mechanistic studies indicate that inhibition of Wee1 abrogates the S-phase checkpoint and augments apoptosis induced by cytarabine. In AML and lung cancer cell lines, genetic disruption of p53 did not alter the cells' enhanced sensitivity to antimetabolites with Wee1 inhibition. Finally, mice with AML were treated with cytarabine and/or MK1775. The combination of MK1775 and cytarabine was well tolerated in mice and enhanced the antileukemia effects of cytarabine, including survival. Thus, inhibition of Wee1 sensitizes hematologic and solid tumor cell lines to antimetabolite chemotherapeutics, whether p53 is functional or not, suggesting that the use of p53 mutation as a predictive biomarker for response to Wee1 inhibition may be restricted to certain cancers and/or chemotherapeutics. These data provide preclinical justification for testing MK1775 and cytarabine in patients with leukemia.

Topics: Animals; Antimetabolites, Antineoplastic; Apoptosis; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Synergism; Female; Humans; Leukemia, Myeloid, Acute; Mice; Nuclear Proteins; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Forodesine and nelarabine (the pro-drug of ara-G) are 2 nucleoside analogues with promising anti-leukemic activity. To better understand which pediatric patients might benefit from forodesine or nelarabine (ara-G) therapy, we investigated the in vitro sensitivity to these drugs in 96 diagnostic pediatric leukemia patient samples and the mRNA expression levels of different enzymes involved in nucleoside metabolism. Forodesine and ara-G cytotoxicities were higher in T-cell acute lymphoblastic leukemia (T-ALL) samples than in B-cell precursor (BCP)-ALL and acute myeloid leukemia (AML) samples. Resistance to forodesine did not preclude ara-G sensitivity and vice versa, indicating that both drugs rely on different resistance mechanisms. Differences in sensitivity could be partly explained by significantly higher accumulation of intracellular dGTP in forodesine-sensitive samples compared with resistant samples, and higher mRNA levels of dGK but not dCK. The mRNA levels of the transporters ENT1 and ENT2 were higher in ara-G-sensitive than -resistant samples. We conclude that especially T-ALL, but also BCP-ALL, pediatric patients may benefit from forodesine or nelarabine (ara-G) treatment.

Topics: Antineoplastic Agents; Arabinonucleosides; Cell Line, Tumor; Child; Deoxycytidine Kinase; Deoxyguanine Nucleotides; Drug Resistance, Neoplasm; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Gene Expression; Humans; In Vitro Techniques; Leukemia, Myeloid, Acute; Leukemia, Prolymphocytic, B-Cell; Phosphotransferases (Alcohol Group Acceptor); Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Prodrugs; Purine Nucleosides; Purines; Pyrimidinones; RNA, Messenger; RNA, Neoplasm

Subcellular localization of photosensitizers is thought to play a critical role in determining the mode of cell death after photodynamic treatment (PDT) of leukemia cells. Using confocal laser scanning microscopy and fluorescent organelle probes, we examined the subcellular localization of merocyanine 540 (MC540) in the murine myeloid leukemia M1 and WEHI 3B (JCS) cells. Two patterns of localization were observed: in JCS cells, MC540 was found to localize on the plasma membrane and mitochondria; and in M1 leukemia cells, MC540 was found to localize on lysosomes. The relationship between subcellular localization of MC540 and PDT-induced apoptosis was investigated. Apoptotic cell death, as judged by the formation of apoptotic nuclei, was observed 4 h after irradiation in both leukemia cell lines. Typical ladders of apoptotic DNA fragments were also detected by DNA gel electrophoresis in PDT-treated JCS and M1 cells. At the irradiation dose of 46 kJ/m2 (LD90 for JCS and LD86 for M1 cells), the percentage of apoptotic JCS and M1 cells was 78 and 38%, respectively. This study provided substantial evidence that MC540 localized differentially in the mitochondria, and the subsequent photodamage of the organelle played an important role in PDT-mediated apoptosis in myeloid leukemia cells.

Topics: Animals; Apoptosis; Leukemia, Myeloid, Acute; Mice; Mitochondria; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Subcellular Fractions; Tumor Cells, Cultured

The effect of merocyanine 540 (Mc 540) mediated photoirradiation on both neoplastic and normal hemopoietic progenitor cells was studied. Bone marrow (BM) cells from children with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) at initial diagnosis, ALL in remission, neuroblastoma and normal children as well as cells of Reh-6 and HL-60 cell lines were incubated with Mc 540 in the presence of human albumin (HA) and exposed to different argon laser 514 nm doses. Cell survival was estimated using Trypan Blue supravital stain following a 24-h incubation and leukemic cell lines were studied in continuous cell cultures of 4 weeks duration. Our results showed that HA protects normal BM cells from Mc 540 mediated phototoxicity. A 99.9999% inhibition of Reh-6 and HL-60 was noted at irradiation doses where the corresponding mean survival of normal BM cells was 77.4 +/- 12 and 70.3 +/- 10%, respectively. BM leukemic cells from children with ALL and AML were also very sensitive to Mc 540 photoirradiation in contrast to neuroblastoma cells where only a three-fold reduction was observed. Finally, the survival of normal BM progenitors was 38% for colony forming unit erythroid CFU-E, 37% for burst forming unit erythroid BFU-E, 55% for CFU-GM and 29% for CFU-GEMM. In conclusion it seems that Mc 540 mediated photoirradiation in neoplastic cells exerts selective cytotoxicity and can be used in ex vivo purging of malignant cells in BM.

Topics: Bone Marrow; Bone Marrow Cells; Bone Marrow Purging; Cell Line; Cell Survival; Child; Dose-Response Relationship, Radiation; Hematopoietic Stem Cells; HL-60 Cells; Humans; Lasers; Leukemia, Myeloid, Acute; Neuroblastoma; Photolysis; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Pyrimidinones; Radiation-Sensitizing Agents

In a model for ex-vivo purging of bone marrow grafts, leukemic cells and normal bone marrow cells were treated with merocyanine 540 and exposed to 514 nm laser light. With this treatment, 99.9999% of leukemic cells were killed while 55% of the normal bone marrow cells survived. The deleterious effects of laser light alone in the absence of photosensitizer were not observed as determined by cell viability, cell migration, and response of target cells to human migration inhibition factor. These results indicate that laser light induced photodynamic therapy can be useful for ex-vivo autologous bone marrow purging without regard to the deleterious effects of laser light alone.

Topics: Bone Marrow; Bone Marrow Cells; Cell Line; Cell Migration Inhibition; Cell Survival; Humans; Lasers; Leukemia, Myeloid, Acute; Macrophage Migration-Inhibitory Factors; Macrophages; Photochemotherapy; Pyrimidinones; Tumor Cells, Cultured

We studied the effects of 514-nm laser light-induced merocyanine 540 (MC540)-mediated toxicity on both leukemic and normal bone marrow (BM) cells. Acute promyelocytic leukemia (HL-60) cells were incubated with MC540 (20 micrograms/ml) and exposed to 93.6 J/cm2 irradiation at a 514-nm wavelength. Normal bone marrow cells were treated under similar conditions. At this dose, 99.9999% of the leukemic cells were killed while 55% of the BM cell survived. Of the granulocyte-macrophage colony-forming cells (CFU-GM), 27% also survived this treatment. Photosensitization of a mixture of irradiated BM cells mixed with an equal number of nonirradiated HL-60 cell did not interfere with the killing of HL-60 cells. There was no significant reduction in the viability of cells when exposed to the laser light alone. In summary, laser light-induced photosensitization with MC540 has a selective cytotoxicity to leukemic cells; therefore, this procedure may be useful for purging neoplastic cells from autologous BM.

Topics: Bone Marrow; Bone Marrow Cells; Cell Survival; Colony-Forming Units Assay; Dose-Response Relationship, Radiation; Granulocytes; Humans; Laser Therapy; Leukemia, Myeloid, Acute; Macrophages; Photochemotherapy; Pyrimidinones; Radiation-Sensitizing Agents; Stem Cells; Tumor Cells, Cultured

Human myeloid leukemia KG-1 cells are induced to differentiate to macrophage-like cells by tumor-promoting phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA). Cells from the cloned subline, KG-1a, unlike the parental line, are resistant to the differentiating effect of TPA. In the present studies, we investigated in these cells protein phosphorylation stimulated by various protein kinase C activators, including 1-oleoyl-2-acetylglycerol in the presence of the diacylglycerol kinase inhibitor R59022, TPA, mezerein, and bryostatin. All the agents stimulated, to a greater extent and with a higher potency, phosphorylation of several proteins in KG-1 cells than in KG-1a cells. On the other hand, these agents markedly stimulated phosphorylation of other proteins in KG-1a cells compared to that in KG-1 cells. The findings indicated that the actions of the diacylglycerol, 1-oleoyl-2-acetylglycerol, and the non-metabolizable activators (TPA, mezerein, and bryostatin) were very similar but not fully equivalent; and that KG-1a cells exhibited altered (increased or decreased) phosphorylation patterns, perhaps related to the TPA resistance characteristic of this subline of cells.

Topics: Bryostatins; Cell Differentiation; Cell Line; Diglycerides; Diterpenes; Drug Resistance; Enzyme Activation; Humans; Lactones; Leukemia, Myeloid, Acute; Macrolides; Phosphorylation; Protein Kinase C; Proteins; Pyrimidinones; Terpenes; Tetradecanoylphorbol Acetate; Thiazoles

To stimulate a leukemia remission marrow, cell suspensions of normal human bone marrow were mixed with human acute lymphoblastic or myelogenous leukemic cells of the CCRF-SF, Nalm-6, and K-562 lines. The cell mixtures were incubated in vitro with mafosfamide (AZ) or with the photoreactive dye merocyanine 540 (MC-540). A quantity of 10(4) cells of the treated suspensions was dispensed into microculture plates, and graded cell numbers of the line used to contaminate the normal marrow were added. Limiting-dilution analysis was used to estimate the frequency of leukemia cells persisting after treatment with the decontaminating agents. Treatment with AZ or MC-540 produced a total elimination (ie, 6 logs or 5.3 logs respectively) of B cell acute leukemia cells (CCRF-SB), whereas nearly 1.7 logs and 2 logs of K-562 acute myelogenous blasts were still present in the cell mixtures after treatment with MC-540 and AZ, respectively. Treatment of the Nalm-6-contaminated cell mixtures with AZ resulted in 100% elimination of clonogenic cells, whereas nearly 80% decontamination was obtained with MC-540. Our results suggest that treatment with AZ could be an effective method of eliminating clonogenic tumor cells from human bone marrow. MC-540, shown by previous studies to spare sufficient pluripotential stem cells to ensure hemopoietic reconstitution in the murine model and in clinical application, has comparable effects and merits trials for possible clinical use in autologous bone marrow transplantation.

Topics: Bone Marrow; Bone Marrow Cells; Cell Line; Cyclophosphamide; Humans; Leukemia, Lymphoid; Leukemia, Myeloid, Acute; Pyrimidinones; Radiation-Sensitizing Agents

After fixation in a modified Bouin's solution, the acid dye merocyanine 540 stained granules in granulocytic cells intensely. In immature granulocytes, such as promyelocytes and myelocytes, granules stained pink to violet. In some leukemic myeloblasts, promyelocytes and monocytes, granules also stained deep pink to violet. In more mature granulocytes, such as metamyelocytes, bands, and neutrophils, granules stained bright red to orange. In eosinophils and basophils, granules stained deep red. Granules of the type described were not visualized in normal plasma cells, lymphocytes, monocytes, or megakaryocytes. In normoblasts, cytoplasm stained diffusely red. Cytoplasmic staining in erythroblasts became darker as the cell matured, probably reflecting hemoglobin content. Used as a single agent stain, merocyanine 540 may be useful in distinguishing normal and leukemic granulocytic cells from other types of blood cells.

Topics: Bone Marrow; Bone Marrow Cells; Granulocytes; Humans; Leukemia; Leukemia, Lymphoid; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Pyrimidinones; Reference Values; Staining and Labeling