Growth Inhibition Of Leukemia Cells Research Articles

Phase 2 Trial of Single Agent Gedatolisib (PF-05212384), a Dual PI3K/mTOR Inhibitor, for Adverse Prognosis and Relapse/Refractory AML: Clinical and Transcriptomic Results

Background: Acute Myeloid Leukemia (AML) is a heterogeneous disease carrying a dismal prognosis. This outcome is the consequence of patient factors (age, comorbidity) and intrinsic biologic abnormalities. Cytogenetic and mutational data are already used to define treatment strategy, but many cellular signaling pathways are deregulated in AML, including targetable PI3K/mTOR pathway. Activation of the PI3K/mTOR pathway promotes cell growth, survival and resistance to chemotherapy and is detected in virtually all AML samples. mTOR, a serine/threonine kinase downstream of PI3K, may be activated independently of PI3K. Gedatolisib is a small molecule in development of solid tumors. It inhibits both PI3K and mTOR kinases, which may result in apoptosis and growth inhibition of leukemic cells. We explored the clinical activity and tolerance of Gedatolisib in the particularly underserved group of relapse/refractory and adverse prognosis AML.Methods: We conducted an open, prospective, single-arm, multicentric phase 2 trial in 4 academic French hematology centers. Enrollment took place between September 2015 and August 2016. Inclusion criteria were relapse/refractory AML and therapy-related AML. Experimental drug dosage was derived from phase 1 studies in solid tumors. Treatment protocol consisted in 4 cycles, each cycle containing 4 weekly injections of Gedatolisib 150 mg delivered IV. Monitoring included weekly blood counts and bone marrow (BM) aspiration after 2 and 4 cycles. Primary objective was Overall Response Rate and secondary objectives were Progression-Free Survival (PFS) and Overall Survival (OS). An ancillary biologic study focused on RNA sequencing (RNAseq) of AML-infiltrated BM samples (>30% blasts at diagnosis) before and after drug exposure to explore its molecular impact. This trial was funded by French National Cancer Institute (INCa) and registered as NCT02438761.Results: 12 patients were included but only 10 patients were retained in the final analysis. 6 patients (pts) were males (60%) and the median age was 59.6 years (range: 33-74). All pts had relapsed AML (5 pts in 1st relapse, 5 pts in 2nd relapse) and symptomatic uncontrolled AML. 1 patient (pt) had therapy-related AML (prostate adenocarcinoma). Cytogenetic risk: 4 pts had intermediate and 6 pts had adverse risk. Only 1 pt completed the treatment including 4 cycles of Gedatolisib. 1 pt received 3 cycles, 3 pts received 2 cycles and 5 pts received 1 cycle (i.e. 4 injections). Reasons for early patient withdrawal were: objective disease progression for 5 pts (including uncontrolled hyperleukocytosis for 4 pts), global deterioration of health status for 2 pts, treatment-related adverse event for 1 pt (anal ulcer), treatment-unrelated adverse event for 1 patient (pneumonia). No objective response was detected for any of the 10 pts according to the IWG AML criteria (Cheson, 2003). Serious adverse events included mouth ulcers (2 pts) and anal ulcer (1 pt). Median follow-up was 4.4 months (mo) (range: 1.7-16.3). OS was 3.5 mo (IC95%=2 mo). PFS was 2.3 mo (IC95%=2 mo). The biologic study used 6 paired BM samples (at inclusion and after drug exposure) which could be analyzed by RNAseq: 1 pair after 4 cycles, 3 pairs after 2 cycles, 2 pairs after more than 1 cycle (6 and 7 drug injections). Transcriptomic principal component analysis showed that each pair clustered together and apart of the other samples, reflecting the heterogeneity of the cohort. The drug impacts weakly gene expression pattern as only 38 genes were significantly differentially-expressed. Among up-regulated genes, 4 were associated with NK cell immunity. Gedatolisib might affect immune cells as already described for other PI3K inhibitors. We detected 23 pathogenic variants and 58 variants of unknown pathogenicity (median=6 variants/sample [1-22]) without obvious correlation with drug resistance. Ongoing research focuses on genes and pathways previously associated with PI3K inhibitor resistance in solid tumors.Conclusion: Gedatolisib, a dual PI3K/mTOR inhibitor used as a single therapy had no clinical benefit in adverse prognosis and relapsed/refractory AML patients. Combination of Gedatolisib with chemotherapy was not explored, yet this could be a potential therapeutic development, as it was recently shown for Midostaurin and FLT3-mutated AML. DisclosuresNo relevant conflicts of interest to declare.

1,3,4-oxadiazole/chalcone hybrids: Design, synthesis, and inhibition of leukemia cell growth and EGFR, Src, IL-6 and STAT3 activities

A new series of 1,3,4-oxadiazole/chalcone hybrids was designed, synthesized, identified with different spectroscopic techniques and biologically evaluated as inhibitors of EGFR, Src, and IL-6. The synthesized compounds showed promising anticancer activity, particularly against leukemia, with 8v being the most potent. The synthesized compounds exhibited strong to moderate cytotoxic activities against K-562, KG-1a, and Jurkat leukemia cell lines in MTT assays. Compound 8v showed the strongest cytotoxic activity with IC50 of 1.95 µM, 2.36 µM and 3.45 µM against K-562, Jurkat and KG-1a leukemia cell lines, respectively. Moreover; the synthesized compounds inhibited EGFR, Src, and IL-6. Compound 8v was most effective at inhibiting EGFR (IC50 = 0.24 μM), Src (IC50 = 0.96 μM), and IL-6 (% of control = 20%). Additionally, most of the compounds decreased STAT3 activation.

Deregulation of linc-PINT in acute lymphoblastic leukemia is implicated in abnormal proliferation of leukemic cells.

Long Non-Coding RNAs (lncRNAs) are functional RNAs longer than 200 nucleotides in length. Several lncRNAs are involved in cell proliferation and are deregulated in several human tumors. Few lncRNAs have been described to play a role in Acute Lymphoblastic Leukemia (ALL). In this study, we carried out a genome wide lncRNA expression profiling in ALL samples and peripheral blood samples obtained from healthy donors. We detected 43 lncRNAs that were aberrantly expressed in ALL. Interestingly, among them, linc-PINT showed a significant downregulation in T and B-ALL. Re-expression of linc-PINT in ALL cells induced inhibition of leukemic cell growth that was associated with apoptosis induction and cell cycle arrest in G2/M phase. linc-PINT induced the transcription of HMOX1 which reduced the viability of ALL cells. Intriguingly, we observed that treatment with anti-tumoral epigenetic drugs like LBH-589 (Panobinostat) and Curcumin induced the expression of linc-PINT and HMOX1 in ALL. These results indicate that the downregulation of linc-PINT plays a relevant role in the pathogenesis of ALL, and linc-PINT re-expression may be one of the mechanisms exerted by epigenetic drugs to reduce cell proliferation in ALL.

Ceritinib Enhances the Efficacy of Substrate Chemotherapeutic Agent in Human ABCB1-Overexpressing Leukemia Cells In Vitro, In Vivo and Ex-Vivo

Background/Aims: Multidrug resistance (MDR) triggered by ATP binding cassette (ABC) transporters, such as ABCB1, ABCC1, and ABCG2, is a key obstacle for successful cancer chemotherapy. There is currently no FDA-approved MDR modulator that can be used in clinic. Ceritinib, a selective ALK inhibitor, has been approved as the second-line treatment for ALK-positive non-small cell lung cancer. Here, we examined the role of ceritinib in leukemia associated MDR in therapy. Methods: The cell proliferation was detected by MTT assay. The flow cytometry was used to detect the expression of cell surface protein and to detect the accumulation and efflux of rhodamine 123 (Rh123) or doxorubicin (Dox) in cells. The RT-PCR and Western blot were performed to detect the gene expression and protein expression levels, respectively. Results: We found that ceritinib enhanced the efficacy of substrate chemotherapeutic agent in ABCB1-overexpressing K562/adr leukemia cells both in vitro and in vivo models, but neither in sensitive parental K562 leukemia cells nor in ABCC1-overexpressing HL-60/adr leukemia cells. Mechanistically, ceritinib significantly increased the intracellular accumulation of Rh123 or Dox but did neither alter ABCB1 expressions at both protein and mRNA levels nor block the phosphorylations of AKT and ERK1/2 at the concentration of MDR reversal. Importantly, ceritinib also increased the intracellular accumulation of Dox and enhanced the efficacy of Dox in primary leukemia cells in ex-vivo. Conclusion: Our results suggested that ceritinib enhanced the efficacy of substrate chemotherapeutic agent on inhibition of leukemia cell growth in vitro, in vivo and ex-vivo, which linked to block ABCB1 function, pumping out its substrate conventional chemotherapeutic agent, thereby increasing the intracellular accumulation. These suggest the combination of ceritinib and substrate chemotherapeutic drugs maybe an effective treatment of resistant leukemia patients with ABCB1-mediated MDR.

Peroxoniobium inhibits leukemia cell growth.

A new class of polyoxoniobate complex has been synthesized and characterized as a novel anticancer agent for photodynamic therapy. The complex inhibits the growth of chronic myelogenous leukemia cells with an IC50 value of 30 μM, in the dark. However, upon exposure to light (365 nm) there is a fivefold increase in the cytotoxic activity. Light radiation activate the complex with the formation of radical species capable of interacting with DNA according to our experimental and theoretical data.

Matrine inhibits BCR/ABL mediated ERK/MAPK pathway in human leukemia cells.

The BCR/ABL fusion gene and its downstream signaling pathways such as Ras/Raf/MAPK, JAK/STAT3, and PI3K/AKT pathways play important roles in malignant transformation of leukemia, especially chronic myelogenous leukemia (CML). Our previous study showed that matrine, an alkaloid extracted from a Chinese herb radix sophorae, significantly inhibited the proliferation of human CML K562cells, induced cell cycle arrest in G0/G1, and promoted cell apoptosis. In the present study, we investigated the molecular mechanism of matrine in the growth inhibition of leukemia cells using K562 and HL-60 cell lines. RT-PCR and Western blot assay demonstrated that the expression of BCR/ABL in K562 and HL-60 cells was significantly inhibited by matrine treatment. Phosphorylation of MEK1, ERK1/2, and their upstream adaptor molecules Shc and SHP2 were significantly downregulated. The protein and mRNA expression of components of the ERK/MAPK signal pathway, and Bcl-xL, Cyclin D1, and c-Myc, were dramatically reduced. Conversely, the expression of p27, a negative regulator of cell cycle progression, increased after matrine treatment. These results indicated that the inhibition of ERK/MAPK and BCR/ABL signaling pathway was associated with matrine’s suppressive effects on the growth of K562 and HL-60 cells. In in vivo study, matrine significantly decreased the mortality rate of tumor-baring mice and suggested that matrine could exert its anti-leukemia effect in vivo.

Jab1/Csn5-Thioredoxin Signaling in Relapsed Acute Monocytic Leukemia under Oxidative Stress.

Purpose: High levels of ROS and ineffective antioxidant systems contribute to oxidative stress, which affects the function of hematopoietic cells in acute myeloid leukemia (AML); however, the mechanisms by which ROS lead to malignant transformation in relapsed AML-M5 are not completely understood. We hypothesized that alterations in intracellular ROS would trigger AML-M5 relapse by activating the intrinsic pathway.Experimental Design: We studied ROS levels and conducted c-Jun activation domain-binding protein-1 (JAB1/COPS5) and thioredoxin (TRX) gene expression analyses with blood samples obtained from 60 matched AML-M5 patients at diagnosis and relapse and conducted mechanism studies of Jab1's regulation of Trx in leukemia cell lines.Results: Our data showed that increased production of ROS and a low capacity of antioxidant enzymes were characteristics of AML-M5, both at diagnosis and at relapse. Consistently, increased gene expression levels of TRX and JAB1/COPS5 were associated with low overall survival rates in patients with AML-M5. In addition, stimulating AML-M5 cells with low concentrations of hydrogen peroxide led to increased Jab1 and Trx expression. Consistently, transfection of ectopic Jab1 into leukemia cells increased Trx expression, whereas silencing of Jab1 in leukemia cells reduced Trx expression. Mechanistically, Jab1 interacted with Trx and stabilized Trx protein. Moreover, Jab1 transcriptionally regulated Trx. Furthermore, depletion of Jab1 inhibited leukemia cell growth both in vitro and in vivoConclusions: We identified a novel Jab1-Trx axis that is a key cellular process in the pathobiologic characteristics of AML-M5. Targeting the ROS/Jab1/Trx pathway could be beneficial in the treatment of AML-M5. Clin Cancer Res; 23(15); 4450-61. ©2017 AACR.

Thymoquinone exerts potent growth-suppressive activity on leukemia through DNA hypermethylation reversal in leukemia cells.

Thymoquinone (TQ), a bioactive constituent of the volatile oil of Monarda fistulosa and Nigella sativa, possesses cancer-specific growth inhibitory effects, but the underlying molecular mechanisms remain largely elusive. We propose that TQ curbs cancer cell growth through dysfunction of DNA methyltransferase 1 (DNMT1). Molecular docking analysis revealed that TQ might interact with the catalytic pocket of DNMT1 and compete with co-factor SAM/SAH for DNMT1 inhibition. In vitro inhibitory assays showed that TQ decreases DNMT1 methylation activity in a dose-dependent manner with an apparent IC50 of 30 nM. Further, exposure of leukemia cell lines and patient primary cells to TQ resulted in DNMT1 downregulation, mechanistically, through dissociation of Sp1/NFkB complex from DNMT1 promoter. This led to a reduction of DNA methylation, a decrease of colony formation and an increase of cell apoptosis via the activation of caspases. In addition, we developed and validated a sensitive and specific LC-MS/MS method and successfully detected a dynamic change of TQ in mouse plasma after administration of TQ through the tail vein, and determined a tolerable dose of TQ to be 15 mg/kg in mouse. TQ administration into leukemia-bearing mice induced leukemia regression, as indicated by the reversed splenomegaly and the inhibited leukemia cell growth in lungs and livers. Our study for the first time demonstrates that DNMT1-dependent DNA methylation mediates the anticancer actions of TQ, opening a window to develop TQ as a novel DNA hypomethylating agent for leukemia therapy.

The novel BMI-1 inhibitor PTC596 downregulates MCL-1 and induces p53-independent mitochondrial apoptosis in acute myeloid leukemia progenitor cells

Disease recurrence is the major problem in the treatment of acute myeloid leukemia (AML). Relapse is driven by leukemia stem cells, a chemoresistant subpopulation capable of re-establishing disease. Patients with p53 mutant AML are at an extremely high risk of relapse. B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is required for the self-renewal and maintenance of AML stem cells. Here we studied the effects of a novel small molecule inhibitor of BMI-1, PTC596, in AML cells. Treatment with PTC596 reduced MCL-1 expression and triggered several molecular events consistent with induction of mitochondrial apoptosis: loss of mitochondrial membrane potential, BAX conformational change, caspase-3 cleavage and phosphatidylserine externalization. PTC596 induced apoptosis in a p53-independent manner. PTC596 induced apoptosis along with the reduction of MCL-1 and phosphorylated AKT in patient-derived CD34+CD38low/− stem/progenitor cells. Mouse xenograft models demonstrated in vivo anti-leukemia activity of PTC596, which inhibited leukemia cell growth in vivo while sparing normal hematopoietic cells. Our results indicate that PTC596 deserves further evaluation in clinical trials for refractory or relapsed AML patients, especially for those with unfavorable complex karyotype or therapy-related AML that are frequently associated with p53 mutations.

Identification of CHD4 As a Potential Therapeutic Target of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by impaired myeloid differentiation of hematopoietic progenitors, causing uncontrolled proliferation and accumulation of immature myeloid cells in the bone marrow. Rearrangements of the mixed lineage leukemia (MLL) gene are common aberrations in acute leukemia and occur in over 70% in childhood leukemia and 5-10% in leukemia of adults. MLL rearrangements encode a fusion oncogenic H3K4 methytransferase protein, which is sufficient to transform hematopoietic cells and give rise to an aggressive subtype of AML. Leukemia where the MLL fusion oncogene is expressed is characterized by dismal prognosis and 30-60% of 5-years overall survival rate.The current standard treatment for AML is chemotherapy and in certain cases bone marrow transplantation. However, chemotherapy causes severe side effects on normal cells and an increased risk of relapse. Consequently, discovery of novel drug targets with better efficacy and low toxicity are needed to improve treatment of AML.In this study, we aimed to identify genes that are required for growth of AML cells and that encode proteins that potentially could be used as therapeutic targets. To do this, we performed high-throughput RNAi screening covering all annotated human genes and the homologous genes in mice, using barcoded lentiviral-based shRNA vectors. Stable loss-of-function screening was done in three AML cell lines (two human and one murine AML cell lines) as well as in a non-transformed hematopoietic control cell line. The candidate genes were selected based on that shRNA-mediated knockdown caused at least a 5-fold growth inhibition of leukemic cells and that the individual candidates were targeted by multiple shRNAs.The chromodomain Helicase DNA binding protein 4 (CHD4), a chromatin remodeler ATPase, displayed the most significant effect in reduced AML cell proliferation upon inhibition among the overlapping candidate genes in all three AML cell lines. CHD4 is a main subunit of the Nucleosome Remodeling Deacetylase (NuRD) complex and has been associated with epigenetic transcriptional repression. A recent study has shown that inhibition of CHD4 sensitized AML cells to genotoxic drugs by chromatin relaxation, which increases rate of double-stranded break (DSB) in leukemic cells.To verify whether CHD4 is exclusively essential for AML with MLL rearrangements, we inhibited CHD4 expression with two independent shRNAs in various AML cell lines with and without MLL translocations. In vitro monitoring of growth and viability indicated that knockdown of CHD4 efficiently suppressed growth in all tested cell lines, suggesting that CHD4 is required in general for growth of leukemic cells. To test the effect of CHD4 inhibition in normal hematopoiesis, we pursued knockdown of CHD4 and monitored effects in hematopoiesis using colony formation assays of human CD34+ cells. The results demonstrated that CHD4 knockdown had minor effects in colony formation as well as growth and survival of normal hematopoietic cells.Furthermore, to explore whether inhibition of CHD4 can prevent AML tumor growth and disease progression in vivo, we have generated a mouse model for AML. By transplanting AML cells transduced with shRNA against CHD4 into recipient mice, we showed that shRNA-mediated targeting of CHD4 not only significantly prolonged survival of AML transplanted mice but also in some cases completely rescued some mice from development of the disease. Collectively, these data suggested that CHD4 is required for AML maintenance in vivo.Next, to determine whether suppression of CHD4 can inhibit cell growth of different subpopulations and subtypes of AML, we performed loss of function studies of CHD4 on patient-derived AML cells ex vivo. Loss of CHD4 expression significantly decreased the frequency of leukemic initiating cells in different subtypes AML patient samples. In further in vivo studies using a xeno-tranplantation model for AML, we demonstrated that shRNA-mediated inhibition of CHD4 significantly reduced the frequency of leukemic cells in the marrow 6 weeks after transplantation.Taken together our results demonstrated the critical and selective role of CHD4 in propagation of patient-derived AML cells as well as in disease progression in mouse models for AML. We believe that CHD4 represents a novel potential therapeutic target that can be used to battle AML. DisclosuresNo relevant conflicts of interest to declare.

The Novel Thiosemicarbazone Derivative Coti-2 Induces Mitochondrial Apoptosis in Acute Myeloid Leukemia Cells Via a p53-Independent Mechanism

IntroductionTP53 mutations are rare (< 5%) in de novo acute myeloid leukemia (AML) but if present, they are associated with a very poor prognosis (< 1% overall survival at 3 years). p53 mutations have been frequently detected in poor-prognosis patients with complex karyotype or therapy-related AML. COTI-2 is a third generation thiosemicarbazone derivative that was identified as a small molecule candidate against a diverse group of human cancer cell lines using a proprietary in silico drug screening method. COTI-2 causes cancer cell death via apoptosis. COTI-2 has been proposed to restore non-functional mutant p53 conformation to functional wild-type conformation in solid cancer cells, and it has recently entered a clinical trial in patients with advanced or recurrent gynecologic malignancies (NCT02433626). The molecular mechanisms of COTI-2 to induce apoptosis, however, remain largely unknown. In this study, we investigated activity and possible modes of action of COTI-2 against AML cells, especially focusing on its p53-independent properties.METHODS AND RESULTS A total of 10 AML cell lines were exposed to COTI-2 for 72 hours and its anti-leukemia effects were determined by viable cell counts and annexin V staining. OCI-AML2, OCI-AML3, MOLM-13, MOLM-14 and MV4;11 express wild-type (WT) p53; THP-1and Kasumi-1 express mutant (MUT) p53; KG-1, U937 and HL-60 does not express p53 protein (p53 NULL). COTI-2 inhibited leukemia cell growth and increased the percentage of annexin V-positive cells, irrespective of cellular p53 status. The IC50 values (concentration at which cell growth is inhibited by 50%) for were not statistically different between p53 WT and MUT/NULL cells (10.3 ± 4.5 nM vs 20.2 ± 11.5 nM, P = 0.44). So did the ED50 values (effective concentration inducing 50% cell killing as measured by Annexin V positivity) (115.0 ± 50.9 nM vs 237.8 ± 109.9 nM, P = 0.34). We next examined the apoptotic effect on primary AML cells from patients with AML. A total of 14 samples [11 p53 WT cases and 3 p53 MUT (C135W, R248Q and C242Y) cases] were examined. COTI-2 induced apoptosis both in p53 WT (40.3 ± 6.4% compound-specific annexin V induction) and MUT samples (48.0 ± 17.7%) (P = 0.62). To elucidate the p53-independent activity of COTI-2, mRNA expression levels of TP53 (p53), CDKN1A (p21) and BBC3 (PUMA) were determined in four AML cell lines (OCI-AML3, MV4;11, HL-60 and Kasumi-1) after exposure to COTI-2. COTI-2 did not induce TP53 or its transcriptional targets CDKN1A or BBC3 in any of the cell lines. To investigate if COTI-2 activates an intrinsic pathway to induce apoptosis in AML, we used p53 NULL HL-60 cells overexpressing BCL-2 (HL-60/BCL-2), BCL-XL (HL-60/BCL-XL) and their controls (HL-60/neo). HL-60/BCL-2 and HL-60/BCL-XL respectively expressed BCL-2 and BCL-XL at greater than 4 times higher levels than HL-60/neo. Interestingly, overexpression of either BCL-2 or BCL-XL almost completely abrogated COTI-2-induced apoptosis. Similar results were obtained in OCI-AML3/BCL-2. MCL-1 overexpression only modestly inhibited COTI-2-induced apoptosis. Involvement of the extrinsic pathway was modest as JurkatI9.2 (a Jurkat clone deficient in caspase-8) and its control showed similar sensitivity to COTI-2. COTI-2 appeared to reduce MCL-1 expression levels through mTORC1 inhibition. Finally, COTI-2 strongly synergized with the BCL-2 inhibitor ABT-199 to induce apoptosis in AML cells.Conclusion COTI-2 induces mitochondrial apoptosis in AML cells, irrespective of their p53 mutational status. COTI-2-induced apoptosis depends on BCL-2 and BCL-XL expression but not on MCL1 expression or p53 activation. COTI-2 has clinical potential in AML that often expresses high levels of MCL-1, especially in combination with BCL-2 inhibition. DisclosuresDanter:Critical Outcomes: Employment.

Pro-inflammatory effect of a traditional Chinese medicine formula with potent anti-cancer activity in vitro impedes tumor inhibitory potential in vivo.

Medicinal formulas are a part of the complex discipline of traditional Chinese medicine that has been used for centuries in China and East Asia. These formulas predominantly consist of the extracts isolated from herbal plants, animal parts and medicinal minerals. The present study aimed to investigate the impact of 150 formulas, used as non-prescription drugs in China, on the treatment of cancer. A formula was identified, C54, commonly used to treat sore throats, which exhibited marked growth inhibition in vitro, associated with cell cycle arrest and apoptosis. Cytotoxicity was, in part, due to the ability of C54 to inhibit the expression and function of the transcription factor, Fli-1, leading to marked inhibition of leukemic cell growth in tissue culture. However, when injected into a model of leukemia initiated by Fli-1 activation, C54 only exhibited a limited tumor inhibition. C54 also did not suppress xenograft growth of the breast cancer cell line, MDA-MB-231, orthopedically transplanted into the mammary fat pad of severe combined immunodeficiency (SCID) mice. Notably, splenomegaly and accumulation of inflammatory CD11b+/Gr1+ monocytes were observed in the tumors and spleens of C54-treated mice. As inflammation is known to accelerate tumor progression, this immune response may counteract the cell-autonomous effect of C54, and account for its limited tumor inhibitory effect in vivo. Combining C54 with an anti-inflammatory drug may improve the potency of C54 for treatment of certain cancers. The present study has highlighted the complexity of Chinese medicinal compounds and the need to thoroughly analyze their systemic effects at high concentrations in vivo.

Effective Concentration of a Multikinase Inhibitor within Bone Marrow Correlates with In Vitro Cell Killing in Therapy-Resistant Chronic Myeloid Leukemia.

Leukemia cells escape BCR-ABL-targeted therapy by developing mutations, such as T315I, in the p210(BCR-ABL) fusion protein in Philadelphia chromosome-positive chronic myeloid leukemia (CML). Although most effort has been focused on development of new tyrosine kinase inhibitors, enrichment of these small-molecule inhibitors in the tumor tissue can also have a profound impact on treatment outcomes. Here, we report that a 2-hour exposure of the T315I-mutant CML cells to 10 μmol/L of the multikinase inhibitor TG101209 suppressed BCR-ABL-independent signaling and caused cell-cycle arrest at G2-M. Further increase in drug concentration to 17.5 μmol/L blocked phosphorylation of the mutant BCR-ABL kinase and its downstream JAK2 and STAT5. The effective dosage to overcome therapy resistance identified in an in vitro setting serves as a guidance to develop the proper drug formulation for in vivo efficacy. A targeted formulation was developed to achieve sustained bone marrow TG101209 concentration at or above 17.5 μmol/L for effective killing of CML cells in vivo Potent inhibition of leukemia cell growth and extended survival were observed in two murine models of CML treated with 40 mg/kg intravenously administered targeted TG101209, but not with the untargeted drug at the same dosage. Our finding provides a unique approach to develop treatments for therapy-resistant CML. Mol Cancer Ther; 15(5); 899-910. ©2016 AACR.

Cutting off the power: inhibition of leukemia cell growth by pausing basal ATP release and P2X receptor signaling?

T cells respond to antigen stimulation with the rapid release of cellular ATP, which stimulates an autocrine feedback mechanism that regulates calcium influx through P2X receptors. This autocrine purinergic feedback mechanism plays an essential role in the activation of T cells resulting in cell proliferation and clonal expansion. We recently reported that increases in mitochondrial ATP production drive this stimulation-induced purinergic signaling mechanism but that low-level mitochondrial ATP production fuels basal T cell functions required to maintain vigilance of unstimulated T cells. Here we studied whether defects in these purinergic signaling mechanisms are involved in the unwanted proliferation of leukemia T cells. We found that acute leukemia T cells (Jurkat) possess a larger number and more active mitochondria than their healthy counterparts. Jurkat cells have higher intracellular ATP concentrations and generat more extracellular ATP than unstimulated T cells from healthy donors. As a result, increased purinergic signaling through P2X1 and P2X7 receptors elevates baseline levels of cytosolic Ca(2+) in Jurkat cells. We found that pharmacological inhibition of this basal purinergic signaling mechanism decreases mitochondrial activity, Ca(2+) signaling, and cell proliferation. Similar results were seen in the leukemic cell lines THP-1, U-937, and HL-60. Combined treatment with inhibitors of P2X1 or P2X7 receptors and the chemotherapeutic agent 6-mercaptopurine completely blocked Jurkat cell proliferation. Our results demonstrate that increased mitochondrial metabolism promotes autocrine purinergic signaling and uncontrolled proliferation of leukemia cells. These findings suggest that deranged purinergic signaling can result in T cell malignancy and that therapeutic targeting aimed at purinergic signaling is a potential strategy to combat T cell leukemia.

Artemisinin-derived dimer ART-838 potently inhibited human acute leukemias, persisted in vivo, and synergized with antileukemic drugs.

Artemisinins, endoperoxide-containing molecules, best known as antimalarials, have potent antineoplastic activity. The established antimalarial, artesunate (AS), and the novel artemisinin-derived trioxane diphenylphosphate dimer 838 (ART-838) inhibited growth of all 23 tested acute leukemia cell lines, reduced cell proliferation and clonogenicity, induced apoptosis, and increased intracellular levels of reactive oxygen species (ROS). ART-838 was 88-fold more potent that AS in vitro, inhibiting all leukemia cell lines at submicromolar concentrations. Both ART-838 and AS cooperated with several established antileukemic drugs and newer kinase inhibitors to inhibit leukemia cell growth. ART-838 had a longer plasma half-life than AS in immunodeficient NOD-SCID-IL2Rgnull (NSG) mice, remaining at effective antileukemic concentrations for >8h. Intermittent cycles of ART-838 inhibited growth of acute leukemia xenografts and primagrafts in NSG mice, at higher potency than AS. Based on these preclinical data, we propose that AS, with its established low toxicity and low cost, and ART-838, with its higher potency and longer persistence in vivo, should be further developed toward integration into antileukemic regimens.

Reducing the serine availability complements the inhibition of the glutamine metabolism to block leukemia cell growth

Leukemia cells are described as a prototype of glucose-consuming cells with a high turnover rate. The role of glutamine in fueling the tricarboxylic acid cycle of leukemia cells was however recently identified confirming its status of major anaplerotic precursor in solid tumors. Here we examined whether glutamine metabolism could represent a therapeutic target in leukemia cells and whether resistance to this strategy could arise. We found that glutamine deprivation inhibited leukemia cell growth but also led to a glucose-independent adaptation maintaining cell survival. A proteomic study revealed that glutamine withdrawal induced the upregulation of phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT), two enzymes of the serine pathway. We further documented that both exogenous and endogenous serine were critical for leukemia cell growth and contributed to cell regrowth following glutamine deprivation. Increase in oxidative stress upon inhibition of glutamine metabolism was identified as the trigger of the upregulation of PHGDH. Finally, we showed that PHGDH silencing in vitro and the use of serine-free diet in vivo inhibited leukemia cell growth, an effect further increased when glutamine metabolism was blocked. In conclusion, this study identified serine as a key pro-survival actor that needs to be handled to sensitize leukemia cells to glutamine-targeting modalities.

Transmembrane TNF-α preferentially expressed by leukemia stem cells and blasts is a potent target for antibody therapy

AbstractTo design an effective antibody therapy to improve clinical outcomes in leukemia, the identification of novel cell surface antigens is needed. Herein, we demonstrate a role for transmembrane tumor necrosis factor-α (tmTNF-α) in leukemia. To characterize tmTNF-α expression in acute leukemia (AL), normal hematopoietic cells, and nonhematopoietic tissues, we used a monoclonal antibody, termed C1, which specifically recognizes the tmTNF-α domain. We found that tmTNF-α was preferentially expressed by AL and leukemia stem cells (LSCs). More abundant expression correlated with poor risk stratification, extramedullary infiltration, and adverse clinical parameters. Moreover, knockdown of tmTNF-α+ expression rendered leukemia cells more sensitive to chemotherapy in vitro and delayed regeneration of leukemia in NOD-SCID mice. Targeting tmTNF-α by C1 resulted in leukemia cell killing via antibody-dependent cell-mediated and complement-dependent cytotoxicity in vitro and inhibited leukemia cell growth in vivo while simultaneously sparing normal hematopoietic cells. Notably, C1 administration impaired the regeneration of leukemia in secondary serial transplantation into NOD-SCID mice. In conclusion, tmTNF-α has a favorable AL- and LSC-associated expression profile and is important for the survival and proliferation of these cells. C1-mediated targeting shows potent anti-LSC activity, indicating that tmTNF-α represents a novel target antigen in AL.

Small-molecule inhibitor of glycogen synthase kinase 3β 6-Bromoindirubin-3-oxime inhibits hematopoietic regeneration in stem cell recipient mice.

Small-molecule inhibitors of glycogen synthase kinase 3β (GSK3β) have demonstrated strong anti-leukemia effects in preclinical studies. Here, we investigated the effect of GSK3β inhibitor 6-Bromoindirubin-3-oxime (BIO) previously shown to inhibit leukemia cell growth in vitro and of animal models on hematopoietic regeneration in recipients of stem cell transplant. BIO administered to immunocompromised mice transplanted with human hematopoietic stem cells inhibited human stem cell engraftment in the bone marrow (BM) and peripheral blood. BIO reduced CD34(+) progenitor cells in the BM, and primitive lymphoid progenitors re-populated host thymus at later stages post-transplant. The development of all T-cell subsets in the thymus was suppressed in BIO-treated mice. Human cell engraftment was gradually restored after discontinuation of BIO treatment; however, T-cell depletion remained until the end of experiment, which correlated with the attenuated thymic function in the host. BIO delayed CD34(+) cell expansion in stroma-supported or cytokine-only cultures. BIO treatment delayed progenitor cell divisions and induced apoptosis in cultures with sub-optimal cytokine support. In addition, BIO inhibited B- and T-cell development in co-cultures with MS5 and OP9-DL1 BM stroma cells, respectively. These data suggest that administration of GKS3β inhibitors may act to delay hematopoietic regeneration in patients who received stem cell transplant.

Novel Targeted Therapy for Precursor B Cell Acute Lymphoblastic Leukemia: Anti-CD22 Antibody-MXD3 Antisense Oligonucleotide Conjugates

Precursor B cell (preB) acute lymphoblastic leukemia (ALL) is the most common cancer in children. Despite aggressive treatment, current approaches for preB ALL have significant limitations with a cure rate of only 30% in certain subtypes. In addition, long-term survivors are at risk for late effects that include development of secondary malignancies and toxicity to visceral organs. Therefore, there is a desperate need for more effective and less toxic therapy. [Display omitted] Previously, we demonstrated that the transcription factor MXD3 is a critical regulator of preB ALL cell proliferation. Knockdown of MXD3 expression in preB ALL cells resulted in cell death as determined by annexin V and caspase assays (Satake et al., British Journal of Haematology, in press). We hypothesize that targeted delivery of an MXD3 therapeutic to preB ALL cells will increase therapy effectiveness and decrease off target effects and toxicity, thus increasing the quality of life.In the current study, we developed an antisense oligonucleotide (ASO) that specifically targets MXD3. Taking advantage of anti-CD22 antibody (aCD22 Ab), which specifically targets B cell ALL, we developed aCD22 Ab-MXD3 ASO conjugates as a potential combined phenotypic and molecularly targeted therapeutic. In in vitro tests, using the Reh ALL cell line, we confirmed successful delivery of the aCD22 Ab-MXD3 ASO compound to leukemia cells. This correlated with MXD3 knockdown at the protein level, and inhibition of leukemia cell growth. Cytotoxicity was tested on anonymized healthy donor normal blood cells, including CD34 positive hematopoietic stem cells (HSCs), B cells, and non-B cells from mobilized peripheral blood mononuclear cells. As expected, cytotoxicity was observed in normal B cells, but CD34 HSCs and non-B cells were unaffected. In combination with conventional preB ALL chemotherapy drugs (vincristine or doxorubin), we observed additive cytotoxic effects of aCD22 Ab-MXD3 ASO conjugates on cells in vitro.We determined the compound effectiveness in pre-clinical xenograft animal models of preB ALL, using both the Reh cell line as well as a primary leukemia sample. Age matched female NOD/SCID/IL2Rg-/- (NSG) mice were randomly assigned to 4 treatment groups: 1) PBS, 2) MXD3 ASO and aCD22 Ab unconjugated at the equivalent dose of the high dose group, 3) aCD22 Ab-ASO conjugates at 0.2mg of Ab/kg/dose, and 4) at 1mg of Ab/kg/dose. Five million leukemia cells were inoculated either intravenously (iv) (Reh) or intra-bone marrow (primary cells) to each mouse. Twenty-four hours following leukemia inoculation, animals started receiving twice weekly iv treatments for 3 weeks. All mice treated with PBS and unconjugated MXD3 ASO & aCD22 Ab, died of leukemia at approximately day 21 in the Reh model and at day 30 in the primary leukemia model (Figure). Animals treated with aCD22 Ab-MXD3 ASO conjugates, either at low or high dose, had significantly prolonged survival both in the Reh (p<0.0084, n=4/group) and primary leukemia (p=0.0001, n=8/group) xenograft models (Log-rank (Mantel-Cox) test). Leukemia-related death was confirmed by necropsy. Harvested leukemia cells were HLA and CD22 positive. During treatment, the mice in all the treatment groups remained healthy and active, and did not lose weight. Toxicity was assessed in the primary leukemia model with weekly CBC and chemistry panels and revealed no significant toxicity.In conclusion, we have demonstrated the therapeutic efficacy of the aCD22 Ab-MXD3 ASO conjugates in preB ALL. This is the first study to demonstrate effective direct ASO-conjugated monoclonal Ab-mediated delivery for the treatment of leukemia or other malignancies. Future studies will focus on the further characterization of the compound, effectiveness of combination therapy with standard chemotherapeutic drugs and toxicological profile. DisclosuresNo relevant conflicts of interest to declare.

A rationally designed nanoparticle for RNA interference therapy in B-lineage lymphoid malignancies

The purposes of the present study were to further evaluate the biologic significance of the CD22ΔE12 molecular lesion and determine if it could serve as a molecular target for RNA interference (RNAi) therapy. We show that both pediatric and adult B-lineage lymphoid malignancies are characterized by a very high incidence of the CD22ΔE12 genetic defect. We provide unprecedented experimental evidence for a previously unrecognized causal link between CD22ΔE12 and aggressive biology of BPL cells by demonstrating that siRNA-mediated knockdown of CD22ΔE12 in primary BPL cells is associated with a marked inhibition of their clonogenicity. These findings provide the preclinical proof-of-concept that siRNA-mediated depletion of CD22ΔE12 may help develop effective treatments for high-risk and relapsed BPL patients who are in urgent need for therapeutic innovations. We also describe a unique polypeptide-based nanoparticle formulation of CD22ΔE12-siRNA as an RNAi therapeutic candidate targeting CD22ΔE12 that is capable of delivering its siRNA cargo into the cytoplasm of leukemia cells causing effective CD22ΔE12 depletion and marked inhibition of leukemic cell growth. Further development and optimization of this nanoparticle or other nanoformulation platforms for CD22ΔE12-siRNA may facilitate the development of an effective therapeutic RNAi strategy against a paradigm shift in therapy of aggressive or chemotherapy-resistant B-lineage lymphoid malignancies.