sirolimus and Precursor-B-Cell-Lymphoblastic-Leukemia-Lymphoma

Sinusoidal obstruction syndrome (SOS) is a potentially life-threatening complication that can be observed after allogeneic hematopoietic cell transplantation (HCT). Inotuzumab ozogamicin is an anti-CD22 monoclonal antibody-drug conjugate that has demonstrated high efficacy in relapsed/refractory (R/R) acute lymphoblastic leukemia (ALL) but is associated with an increased risk of SOS in HCT recipients. Here we aimed to examine the incidence and outcomes of SOS in 47 adult patients with R/R ALL who received inotuzumab therapy and subsequently underwent HCT at our institution. All patients received prophylactic therapy with ursodiol, and continuous low-dose heparin also was administered to patients receiving myeloablative conditioning (MAC). SOS occurred in 12 patients (26%) post-HCT, at a median onset of 11 days (range, 3 to 41 days). SOS was graded as very severe in 50% (n = 6), severe in 25% (n = 3), and mild in 25% (n = 3). All patients diagnosed with SOS received treatment with defibrotide for a median of 21 days (range, 3 to 34 days), with resolution of SOS occurring in 8 patients (67%). Mortality from SOS was 33% (n = 4) and occurred at a median of 10 days from diagnosis (range, 3 to 31 days) in patients graded as very severe (n = 3) or severe (n = 1). There were no significant differences between patients who developed SOS and those who did not develop SOS in the median time from the last dose of inotuzumab to transplantation (46 days versus 53 days; P = .37), use of an MAC regimen (42% versus 49%; P = .75), number of lines of therapy prior to inotuzumab (P = .79), median number of administered cycles of inotuzumab (2 versus 2; P = .14), or receipt of inotuzumab as the last therapy prior to HCT (67% versus 66%; P = 1.0). Sirolimus-based graft-versus-host disease (GVHD) prophylaxis was used more frequently in the SOS group (75% versus 29%; P < .01), but there was no between-group difference in the peak sirolimus level (P = .81) or the median time to peak sirolimus level (7 days versus 3.5 days; P = .39). In univariable analysis, only the use of sirolimus-based GVHD prophylaxis was significantly associated with an increased risk of SOS (hazard ratio [HR], 7.50; 95% confidence interval [CI], 1.7 to 33.6; P < .01). In the SOS group, the 100-day mortality rate was 33% (n = 4), and median overall survival (OS) post-HCT was 4.3 months (range, 0.2 to 57.2 months). In the group without SOS, the 100-day mortality rate was 14% (n = 5) and the median OS post-HCT

Topics: Adult; Burkitt Lymphoma; Graft vs Host Disease; Hepatic Veno-Occlusive Disease; Humans; Inotuzumab Ozogamicin; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Sirolimus

A 7-year-old boy with a history of low-risk acute lymphoblastic leukemia developed multiple intussusceptions shortly after the end of maintenance therapy. Explorative laparotomy showed >10 polyps in the small intestine. Histologic examination revealed intestinal smooth muscle sarcomas associated with Epstein-Barr virus. The patient recovered well after partial cuneiform resection of the largest polyps and treatment with sirolimus. This case report indicates that these tumors may arise even after moderate transient immunosuppression and that association with acute lymphoblastic leukemia is possible although rarely described. We discuss the potential benefit of the mTor/Akt signal inhibitors as treatment for these tumors.

Topics: Child; Epstein-Barr Virus Infections; Herpesvirus 4, Human; Humans; Intestinal Neoplasms; Male; Muscle Neoplasms; Muscle, Smooth; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Sarcoma; Sirolimus

B-precursor acute lymphoblastic leukemia (B-pre ALL) is a malignant disorder characterized by the abnormal proliferation of B-cell progenitors. The prognosis of B-pre ALL has improved in pediatric patients, but the outcome is much less successful in adults. Constitutive activation of the phosphatidylinositol 3-kinase (PI3K), Akt and the mammalian target of rapamycin (mTOR) (PI3K/Akt/mTOR) network is a feature of B-pre ALL, where it strongly influences cell growth and survival. RAD001, a selective mTORC1 inhibitor, has been shown to be cytotoxic against many types of cancer including hematological malignancies. To investigate whether mTORC1 could represent a target in the therapy of B-pre ALL, we treated cell lines and adult patient primary cells with RAD001. We documented that RAD001 decreased cell viability, induced cell cycle arrest in G0/G1 phase and caused apoptosis in B-pre ALL cell lines. Autophagy was also induced, which was important for the RAD001 cytotoxic effect, as downregulation of Beclin-1 reduced drug cytotoxicity. RAD001 strongly synergized with the novel allosteric Akt inhibitor MK-2206 in both cell lines and patient samples. Similar results were obtained with the combination CCI-779 plus GSK 690693. These findings point out that mTORC1 inhibitors, either as a single agent or in combination with Akt inhibitors, could represent a potential therapeutic innovative strategy in B-pre ALL.

Topics: Apoptosis; Autophagy; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Everolimus; Heterocyclic Compounds, 3-Ring; Humans; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Oxadiazoles; Phosphoinositide-3 Kinase Inhibitors; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Rapamycin has been shown to inhibit the growth of leukemic cells via an unknown mechanism. In our current study, we show that rapamycin activates autophagy in pediatric t(1;19) pre-B acute lymphoblastic leukemia (pre-B ALL) cells and thereby inhibits proliferation and induces growth arrest in these cells. Rapamycin was found to downregulate an extensive array of positive cell cycle regulators, reduce the total DNA and RNA levels, and specifically downregulate the gene transcription of DNA pol δ1 and RNA pol II. Furthermore, we show that both rapamycin and starvation caused a downregulation of the DNA pol δ1 and RNA pol II proteins which was reversed by the autophagy inhibitor 3-MA. Consistent with the results of our autophagic flux analysis, confocal microscopy indicated that both rapamycin and starvation cause the colocalization of DNA pol δ1 and RNA pol II with GFP-LC3 at autophagosomes. This colocalization was blocked by the autophagy inhibitor bafilomycin A1 which inhibits the fusion between autophagosomes and lysosomes. These data suggest that rapamycin inhibits the growth of pediatric t(1;19) pre-B ALL cells through both transcriptional inhibition and autophagic degradation of DNA pol δ1 and RNA pol II.

Topics: Autophagy; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; DNA Polymerase III; Down-Regulation; Gene Expression Regulation, Leukemic; HEK293 Cells; Humans; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; RNA Polymerase II; Sirolimus

CRLF2 rearrangements, JAK1/2 point mutations, and JAK2 fusion genes have been identified in Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL), a recently described subtype of pediatric high-risk B-precursor ALL (B-ALL) which exhibits a gene expression profile similar to Ph-positive ALL and has a poor prognosis. Hyperactive JAK/STAT and PI3K/mammalian target of rapamycin (mTOR) signaling is common in this high-risk subset. We, therefore, investigated the efficacy of the JAK inhibitor ruxolitinib and the mTOR inhibitor rapamycin in xenograft models of 8 pediatric B-ALL cases with and without CRLF2 and JAK genomic lesions. Ruxolitinib treatment yielded significantly lower peripheral blast counts compared with vehicle (P < .05) in 6 of 8 human leukemia xenografts and lower splenic blast counts (P < .05) in 8 of 8 samples. Enhanced responses to ruxolitinib were observed in samples harboring JAK-activating lesions and higher levels of STAT5 phosphorylation. Rapamycin controlled leukemia burden in all 8 B-ALL samples. Survival analysis of 2 representative B-ALL xenografts demonstrated prolonged survival with rapamycin treatment compared with vehicle (P < .01). These data demonstrate preclinical in vivo efficacy of ruxolitinib and rapamycin in this high-risk B-ALL subtype, for which novel treatments are urgently needed, and highlight the therapeutic potential of targeted kinase inhibition in Ph-like ALL.

Topics: Acute Disease; Animals; Antineoplastic Agents; Child; Disease Models, Animal; Drug Evaluation, Preclinical; Gene Expression Regulation, Neoplastic; Humans; Immunoglobulins; Janus Kinase 1; Janus Kinase 2; Mice; Molecular Targeted Therapy; Nitriles; Philadelphia Chromosome; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Protein Kinase Inhibitors; Pyrazoles; Pyrimidines; Receptors, Cytokine; Signal Transduction; Sirolimus; STAT5 Transcription Factor; Survival Rate; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Despite incremental improvements in outcomes for patients with acute lymphoblastic leukemia, significant numbers of patients still die from this disease. Mammalian target of rapamycin inhibitors have shown potential in vitro and in vivo as therapeutic agents against a range of tumors including acute lymphoblastic leukemia.. Flow cytometry was used to evaluate drug-induced cell death in acute lymphoblastic leukemia cell lines and patients' samples. Human xenografts in immunocompromised mice were used to assess the in vivo effects of selected combinations. Pharmacological inhibitors and lentiviral small interfering ribonucleic acid knock-down of p53 were used to investigate the mechanism of cell killing involved.. Synergistic interactions between RAD001 and cytotoxic agents were demonstrated in vitro and in vivo, with increased caspase-dependent killing. RAD001 suppressed p53 and p21 responses, while suppression of p53 did not prevent killing, indicating p53 independence. RAD001 and cytotoxic agents activated the JUN N-terminal kinase pathway and the combination further increased JUN N-terminal kinase activation. JUN N-terminal kinase inhibition reduced synergistic cell killing by cytotoxic agents and RAD001 in pre-B acute lymphoblastic leukemia cell lines and patients' samples. Bortezomib and MG132, which activate the JUN N-terminal kinase pathway, also synergized with RAD001 in killing pre-B acute lymphoblastic leukemia cells. Killing was greater when RAD001 was combined with proteasome inhibitors than with cytotoxic drugs.. These observations suggest that combining mammalian target of rapamycin inhibitors with conventional chemotherapy or selected novel agents has the potential to improve clinical responses in patients with pre-B acute lymphoblastic leukemia.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Boronic Acids; Bortezomib; Cell Line, Tumor; Combined Modality Therapy; Everolimus; Humans; Immunosuppressive Agents; Leupeptins; Mice; Mice, Inbred NOD; Mice, SCID; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Prognosis; Protease Inhibitors; Pyrazines; Radiation, Ionizing; Sirolimus; Survival Rate; TOR Serine-Threonine Kinases; Whole-Body Irradiation

Despite activity as single agent cancer therapies, Rapamycin (rapa) and its rapalogs may have their greatest effects when combined with other therapeutic modalities. In addition to direct antitumor activity, rapalogs reverse multiple tumor-intrinsic immune evasion mechanisms. These should facilitate tumor-specific T cell activity, but since rapa directly inhibits effector T cells, this potential immune enhancement is lost. We hypothesized that if T cells were rendered resistant to rapa they could capitalize on its downregulation of tumor immune evasion. We therefore modified T cells with a rapa-resistant mutant of mTor, mTorRR, and directed them to B lymphomas by coexpressing a chimeric antigen receptor (CAR) for CD19 (CAR.CD19-28ζ). T cells expressing transgenic mTorRR from a piggyBac transposon maintain mTor signaling, proliferate in the presence of rapa and retain their cytotoxic function and ability to secrete interferon-γ (IFNγ) after stimulation, effector functions that were inhibited by rapa in control T cells. In combination, rapa and rapa-resistant-CAR.CD19-28ζ-expressing T cells produced greater antitumor activity against Burkitt's lymphoma and pre-B ALL cell lines in vitro than CAR.CD19-28ζ T cells or rapa alone. In conclusion, the combination of rapa and rapa-resistant, CAR.CD19-28ζ-expressing T cells may provide a novel therapy for the treatment of B cell malignancies and other cancers.

Topics: Animals; Antigens, CD19; Apoptosis; B-Lymphocytes; Blotting, Western; Burkitt Lymphoma; Cell Proliferation; Cells, Cultured; Combined Modality Therapy; Drug Resistance, Neoplasm; Female; Flow Cytometry; Humans; Immunosuppressive Agents; Interferon-gamma; Lymphocyte Activation; Mice; Mice, Inbred NOD; Mice, SCID; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Receptors, Antigen; Sirolimus; T-Lymphocytes; TOR Serine-Threonine Kinases

Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy affecting children. Despite significant progress and success in the treatment of ALL, a significant number of children continue to relapse and for them, outcome remains poor. Therefore, the search for novel therapeutic approaches is warranted. The aim of this study was to investigate the AMP activated protein kinase (AMPK) as a potential target in childhood acute lymphoblastic leukemia (ALL) subtypes characterized by non-random translocation signature profiles. We evaluated the effects of the AMPK activator AICAR on cell growth, cell cycle regulators and apoptosis of various childhood ALL cells.. We found that treatment with AICAR inhibited cell proliferation, induced cell cycle arrest in G1-phase, and apoptosis in CCRF-CEM (T-ALL), NALM6 (Bp-ALL), REH (Bp-ALL, TEL/AML1) and SupB15 (Bp-ALL, BCR/ABL) cells. These effects were abolished by treatment with the adenosine kinase inhibitor 5'-iodotubericidin prior to addition of AICAR indicating that AICAR's cytotoxicity is mediated through AMPK activation. Moreover, we determined that growth inhibition exerted by AICAR was associated with activation of p38-MAPK and increased expression of the cell cycle regulators p27 and p53. We also demonstrated that AICAR mediated apoptosis through the mitochondrial pathway as revealed by the release of cytochrome C and cleavage of caspase 9. Additionally, AICAR treatment resulted in phosphorylation of Akt suggesting that activation of the PI3K/Akt pathway may represent a compensatory survival mechanism in response to apoptosis and/or cell cycle arrest. Combined treatment with AICAR and the mTOR inhibitor rapamycin resulted in additive anti-proliferative activity ALL cells.. AICAR-mediated AMPK activation was found to be a proficient cytotoxic agent in ALL cells and the mechanism of its anti-proliferative and apoptotic effect appear to be mediated via activation of p38-MAPK pathway, increased expression of cell cycle inhibitory proteins p27 and p53, and downstream effects on the mTOR pathway, hence exhibiting therapeutic potential as a molecular target for the treatment of childhood ALL. Therefore, activation of AMPK by AICAR represents a novel approach to targeted therapy, and suggests a role for AICAR in combination therapy with inhibitors of the PI3K/Akt/mTOR pathways for the treatment of childhood in ALL.

Topics: Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Antineoplastic Agents; Apoptosis; Cell Division; DNA Replication; Drug Delivery Systems; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Enzyme Activation; G1 Phase; Humans; Imidazoles; Leukemia-Lymphoma, Adult T-Cell; Multienzyme Complexes; Neoplasm Proteins; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Prodrugs; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Pyridines; Ribonucleotides; Sirolimus; Tubercidin; Tumor Cells, Cultured

Understanding the pathogenesis of leukemia in the context of lymphopoiesis may reveal novel therapeutic targets. Previously, we have shown that mTOR inhibitors (MTI) show activity in vitro and in preclinical models of both human and murine precursor B acute lymphoblastic leukemia (pre-B ALL), inhibiting cell proliferation and inducing apoptosis. These MTI-mediated effects can be reversed by interleukin-7 (IL-7), an important regulator of early B-cell development. This observation led us to examine the contribution of signaling via the IL-7Ralpha chain, which is shared by the receptor complexes of IL-7 and thymic stromal-derived lymphopoietin (TSLP). TSLP is closely related to IL-7 and active in lymphopoiesis, but an effect of TSLP on leukemia cells has not been described. We examined the effect of TSLP on pre-B ALL cells and their response to MTIs. Here, we show that TSLP stimulates proliferation of pre-B ALL cell lines. TSLP also partially reverses the effects of MTI on proliferation, apoptosis, and ribosomal protein S6 and 4E-BP1 phosphorylation in cell lines, with similar biological effects seen in some primary human lymphoblast samples. These data show that TSLP can promote survival of pre-B ALL cells and antagonize the effects of MTIs. These findings suggest that IL-7Ralpha chain is responsible for transducing the survival signal that overcomes MTI-mediated growth inhibition in pre-B ALL. Thus, further exploration of the IL-7Ralpha pathway may identify potential therapeutic targets in the treatment of ALL. Our data illustrate that growth-factor-mediated signaling may provide one mechanism of MTI resistance.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antibodies, Monoclonal; Apoptosis; Carrier Proteins; Cell Cycle Proteins; Cell Growth Processes; Cytokines; Drug Interactions; Eukaryotic Initiation Factors; Humans; Interleukin-7; Interleukin-7 Receptor alpha Subunit; Janus Kinase 1; Janus Kinase 3; Mice; Mice, Transgenic; Phosphoproteins; Phosphorylation; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Protein Kinase Inhibitors; Protein Kinases; Recombinant Proteins; Ribosomal Protein S6; Signal Transduction; Sirolimus; STAT5 Transcription Factor; Thymic Stromal Lymphopoietin; TOR Serine-Threonine Kinases

c-MET is believed to be an attractive receptor target for molecular therapeutic inhibition. TPR-MET, a constitutively active oncogenic variant of MET, serves as excellent model for testing c-MET inhibitors. Here, we characterized a small molecule c-MET inhibitor, PHA665752, and tested its cooperation with the mammalian target of rapamycin inhibitor as potential targeted therapy.. The effect of PHA665752 treatment was determined on cell growth, motility and migration, apoptosis, and cell-cycle arrest of TPR-MET-transformed cells. Moreover, the effect of PHA665752 on the phosphorylation on MET, as well as its downstream effectors, p-AKT and p-S6K, was also determined. Finally, growth of TPR-MET-transformed cells was tested in the presence of PHA665752 and rapamycin. H441 non-small cell lung cancer (NSCLC) cells (with activated c-Met) were also tested against both PHA665752 and rapamycin.. PHA665752 specifically inhibited cell growth in BaF3. TPR-MET cells (IC(50) < 0.06 micromol/L), induced apoptosis and cell cycle arrest. Constitutive cell motility and migration of the BaF3. TPR-MET cells was also inhibited. PHA665752 inhibited specific phosphorylation of TPR-MET as well as phosphorylation of downstream targets of the mammalian target of rapamycin pathway. When combined with PHA665752, rapamycin showed cooperative inhibition to reduce growth of BaF3. TPR-MET- and c-MET-expressing H441 NSCLC cells.. PHA665752 is a potent small molecule-selective c-MET inhibitor and is highly active against TPR-MET-transformed cells both biologically and biochemically. PHA665752 is also active against H441 NSCLC cells. The c-MET inhibitor can cooperate with rapamycin in therapeutic inhibition of NSCLC, and in vivo studies of this combination against c-MET expressing cancers would be merited.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Drug Synergism; Drug Therapy, Combination; Humans; Indoles; Lung Neoplasms; Mice; Phosphatidylinositol 3-Kinases; Phosphorylation; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-met; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Sulfones; Tumor Cells, Cultured