Activating point mutations of the RAS gene act as driver mutations for a subset of precursor-B cell acute lymphoblastic leukaemias (pre-B ALL) and represent an ambitious target for therapeutic ...approaches. The X box-binding protein 1 (XBP1), a key regulator of the unfolded protein response (UPR), is critical for pre-B ALL cell survival, and high expression of XBP1 confers poor prognosis in ALL patients. However, the mechanism of XBP1 activation has not yet been elucidated in RAS mutated pre-B ALL. Here, we demonstrate that XBP1 acts as a downstream linchpin of the IL-7 receptor signalling pathway and that pharmacological inhibition or genetic ablation of XBP1 selectively abrogates IL-7 receptor signalling via inhibition of its downstream effectors, JAK1 and STAT5. We show that XBP1 supports malignant cell growth of pre-B NRASG12D ALL cells and that genetic loss of XBP1 consequently leads to cell cycle arrest and apoptosis. Our findings reveal that active XBP1 prevents the cytotoxic effects of a dual PI3K/mTOR pathway inhibitor (BEZ235) in pre-B NRASG12D ALL cells. This implies targeting XBP1 in combination with BEZ235 as a promising new targeted strategy against the oncogenic RAS in NRASG12D-mutated pre-B ALL.
The unfolded protein response (UPR) pathway, a stress-induced signaling cascade emanating from the endoplasmic reticulum (ER), regulates the expression and activity of molecules including BiP ...(HSPA5), IRE1 (ERN1), Blimp-1 (PRDM1), and X-box binding protein 1 (XBP1). These molecules are required for terminal differentiation of B cells into plasma cells and expressed at high levels in plasma cell-derived multiple myeloma. Although these molecules have no known role at early stages of B-cell development, here we show that their expression transiently peaks at the pre–B-cell receptor checkpoint. Inducible, Cre-mediated deletion of Hspa5 , Prdm1 , and Xbp1 consistently induces cellular stress and cell death in normal pre-B cells and in pre–B-cell acute lymphoblastic leukemia (ALL) driven by BCR-ABL1 - and NRAS ᴳ¹²ᴰ oncogenes. Mechanistically, expression and activity of the UPR downstream effector XBP1 is regulated positively by STAT5 and negatively by the B-cell–specific transcriptional repressors BACH2 and BCL6. In two clinical trials for children and adults with ALL, high XBP1 mRNA levels at the time of diagnosis predicted poor outcome. A small molecule inhibitor of ERN1-mediated XBP1 activation induced selective cell death of patient-derived pre-B ALL cells in vitro and significantly prolonged survival of transplant recipient mice in vivo. Collectively, these studies reveal that pre-B ALL cells are uniquely vulnerable to ER stress and identify the UPR pathway and its downstream effector XBP1 as novel therapeutic targets to overcome drug resistance in pre-B ALL.
Background The therapeutic armamentarium in multiple myeloma has been significantly broadened by proteasome inhibitors, highly efficient means in controlling of multiple myeloma. Despite the ...developments of therapeutic regimen in treatment of multiple myeloma, still the complete remission requires a novel therapeutic strategy with significant difference in outcomes. Proteasome inhibitors induce autophagy and ER stress, both pivotal pathways for protein homeostasis. Recent studies showed that the IRE1alpha-XBP1 axis of the unfolded protein response (UPR) is up-regulated in multiple myeloma patients. In addition, XBP1 is crucial for the maintenance of viability of acute lymphoblastic leukemia (ALL). Results We analyzed the efficacy of targeting IRE1alpha-XBP1 axis and autophagy in combination with proteasome inhibitor, ixazomib in treatment of multiple myeloma. In this present study, we first show that targeting the IRE1alpha-XBP1 axis with small molecule inhibitors (STF-083010, A106) together with the ixazomib induces cell cycle arrest with an additive cytotoxic effect in multiple myeloma. Further, we examined the efficacy of autophagy inhibitors (bafilomycin A, BAF and chloroquine, CQ) together with ixazomib in multiple myeloma and observed that this combination treatment synergistically reduced cell viability in multiple myeloma cell lines (viable cells Ixa: 51.8 + or - 3.3, Ixa + BAF: 18.3 + or - 7.2, Ixa + CQ: 38.4 + or - 3.7) and patient-derived multiple myeloma cells (Ixa: 59.6 + or - 4.4, Ixa + CQ: 7.0 + or - 2.1). We observed, however, that this combined strategy leads to activation of stress-induced c-Jun N-terminal kinase (JNK). Cytotoxicity mediated by combined proteasome and autophagy inhibition was reversed by addition of the specific JNK inhibitor JNK-In-8 (viable cells: Ixa + BAF: 11.6 + or - 7.0, Ixa + BAF + JNK-In-8: 30.9 + or - 6.1). Conclusion In this study we showed that combined inhibition of autophagy and the proteasome synergistically induces cell death in multiple myeloma. Hence, we consider the implication of pharmaceutical inhibition of autophagy together with proteasome inhibition and UPR-directed therapy as promising novel in vitro treatment strategy against multiple myeloma. Keywords: Autophagy, Multiple myeloma, Proteasome inhibition, Jnk
We report that t(1;19) ALL cells universally exhibit expression of and dependence on the cell surface receptor ROR1. We further identify t(1;19) ALL cell sensitivity to the kinase inhibitor dasatinib ...due to its inhibition of the pre-B cell receptor (pre-BCR) signaling complex. These phenotypes are a consequence of developmental arrest at an intermediate/late stage of B-lineage maturation. Additionally, inhibition of pre-BCR signaling induces further ROR1 upregulation, and we identify distinct ROR1 and pre-BCR downstream signaling pathways that are modulated in a counterbalancing manner—both leading to AKT phosphorylation. Consistent with this, AKT phosphorylation is transiently eliminated after dasatinib treatment, but is partially restored following dasatinib potentiation of ROR1 expression. Consequently, ROR1 silencing accentuates dasatinib killing of t(1;19) ALL cells.
► ROR1 and the pre-BCR are therapeutic targets for t(1;19)-ALL cells ► t(1;19)-ALL cells are universally sensitive to dasatinib because of pre-BCR inhibition ► ROR1 and the pre-BCR drive cooperative signaling cascades ► Silencing of ROR1 accentuates dasatinib killing of t(1;19)-ALL cells
Imatinib has revolutionized the treatment of
B
cr-A
bl1
+
chronic myeloid leukemia (CML), but, in most patients, some leukemia cells persist despite continued therapy, while others become resistant. ...Here, we report that PlGF levels are elevated in CML and that PlGF produced by bone marrow stromal cells (BMSCs) aggravates disease severity. CML cells foster a soil for their own growth by inducing BMSCs to upregulate PlGF, which not only stimulates BM angiogenesis, but also promotes CML proliferation and metabolism, in part independently of
B
cr-A
bl1
signaling. Anti-PlGF treatment prolongs survival of imatinib-sensitive and -resistant CML mice and adds to the anti-CML activity of imatinib. These results may warrant further investigation of the therapeutic potential of PlGF inhibition for (imatinib-resistant) CML.
► PlGF levels are elevated in human and murine CML ► Stromal cell-derived PlGF is upregulated through leukemia cell contact ► PlGF is a mitogen for leukemia cells and stimulates BM angiogenesis ► Anti-PlGF prolongs survival of imatinib-sensitive and -resistant CML mice
Acute lymphoblastic leukemia (ALL) is a disease of lymphoid progenitor cells with an often aggressive course and is commonly caused by the BCR-ABL fusion gene t(9;22) in adults. This fusion gene ...encodes a constitutively active tyrosine kinase that can be effectively inhibited by tyrosine kinase inhibitors (TKIs), with imatinib being the paradigmatic agent of this class. However, BCR-ABL+ ALL cells rapidly develop mutations against many of the available TKIs, and consecutive disease relapse still results in an overall unfavorable prognosis for patients with this disease. To date, allogeneic stem cell transplantation is the only known curative therapeutic option for the mostly elderly patients with BCR-ABL+ ALL. The discrepancy between the limited therapeutic armamentarium and the growing therapeutic need in an aging population is therefore a reason to test drug combinations against BCR-ABL+ ALL. In this study, we demonstrate that the combination of TKIs with proteasome inhibitors efficiently and under certain conditions synergistically exerts cytotoxic effects in BCR-ABL+ ALL cells in vitro with respect to the induction of apoptosis. Both sole and combined treatment of BCR-ABL+ ALL with the proteasome inhibitors bortezomib and ixazomib, respectively, and TKI causes a significantly greater reduction in cell viability than TKI treatment alone in both BCR-ABL+ cell lines TOM-1 and BV-173. In BV-173 cells, we observed a significant reduction in cell viability to only 1.26%±0.46% with bortezomib treatment and 1.57±0.7% with combination treatment, whereas cells treated with dasatinib alone still had a viable percentage of 40.58±2.6%. Similar results were obtained when ixazomib was applied to both cell lines, and apoptosis was induced in both cases (93.36%±2.7% apoptotic BV-173 cells when treated with ixazomib and TKI). The combination of TKI and proteasome inhibitor is efficient in vitro, potentially expanding the spectrum of therapeutic options for patients with BCR-ABL+ ALL.
Here we present the 3-year results of ZUMA-4, a phase I/II multicenter study evaluating the safety and efficacy of KTEX19, an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy, in ...pediatric/adolescent patients with relapsed/refractory B-cell acute lymphoblastic leukemia. Phase I explored two dose levels and formulations. The primary endpoint was the incidence of dose-limiting toxicities. Thirty-one patients were enrolled; KTE-X19 was administered to 24 patients (median age 13.5 years, range 3-20; median follow-up 36.1 months). No dose-limiting toxicities were observed. All treated patients had grade ≥3 adverse events, commonly hypotension (50%) and anemia (42%). Grade 3 cytokine release syndrome rates were 33% in all treated patients, 75% in patients given the dose of 2×106 CAR T cells/kg, 27% in patients given the dose of 1×106 cells/kg in the 68 mL formulation, and 22% in patients given the dose of 1×106 cells/kg in the 40 mL formulation; the percentages of patients experiencing grade ≥3 neurologic events were 21%, 25%, 27%, and 11% respectively. Overall complete remission rates (including complete remission with incomplete hematologic recovery) were 67% in all treated patients, 75% in patients given 2×106 CAR T cells/kg, 64% in patients given 1×106 cells/kg in the 68 mL formulation, and 67% in patients given 1×106 cells/kg in the 40 mL formulation. Overall minimal residual diseasenegativity rates were 100% among responders; 88% of responders underwent subsequent allogeneic stem-cell transplantation. In the 1×106 (40 mL) group (recommended phase II dose), the median duration of remission censored at allogeneic stem-cell transplantation and median overall survival were not reached. Pediatric/adolescent patients with relapsed/refractory B-cell acute lymphoblastic leukemia achieved high minimal residual disease-negative remission rates with a manageable safety profile after a single dose of KTE-X19. Phase II of the study is ongoing at the dose of 1×106 CAR T cells/kg in the 40 mL formulation. ClinicalTrials.gov: NCT02625480.
The adapter protein metastasis suppressor 1 (MTSS1) is implicated as a tumor suppressor or tumor promoter, depending on the type of solid cancer. Here, we identified Mtss1 expression to be increased ...in AML subsets with favorable outcome, while suppressed in high risk AML patients. High expression of MTSS1 predicted better clinical outcome of patients with normal-karyotype AML. Mechanistically, MTSS1 expression was negatively regulated by FLT3-ITD signaling but enhanced by the AML1-ETO fusion protein. DNMT3B, a negative regulator of MTSS1, showed strong binding to the MTSS1 promoter in PML-RARA positive but not AML1-ETO positive cells, suggesting that AML1-ETO leads to derepression of MTSS1. Pharmacological treatment of AML cell lines carrying the FLT3-ITD mutation with the specific FLT3 inhibitor PKC-412 caused upregulation of MTSS1. Moreover, treatment of acute promyelocytic cells (APL) with all-trans retinoic acid (ATRA) increased MTSS1 mRNA levels. Taken together, our findings suggest that MTSS1 might have a context-dependent function and could act as a tumor suppressor, which is pharmacologically targetable in AML patients.
Abstract
BCR-ABL1-positive acute lymphoblastic leukemia (ALL) cell survival is dependent on the inositol-requiring enzyme 1 alpha (IRE1α) branch of the unfolded protein response. In the current ...study, we have focused on exploring the efficacy of a simultaneous pharmacological inhibition of BCR-ABL1 and IRE1α in Philadelphia-positive (Ph+) ALL using tyrosine kinase inhibitor (TKI) nilotinib and the IRE1α inhibitor MKC-8866. The combination of 0.5 µM nilotinib and 30 µM MKC-8866 in Ph+ ALL cell lines led to a synergistic effect on cell viability. To mimic this dual inhibition on a genetic level, pre-B-cells from conditional Xbp1+/fl mice were transduced with a BCR-ABL1 construct and with either tamoxifen-inducible cre or empty vector. Cells showed a significant sensitization to the effect of TKIs after the induction of the heterozygous deletion. Finally, we performed a phosphoproteomic analysis on Ph+ ALL cell lines treated with the combination of nilotinib and MKC-8866 to identify potential targets involved in their synergistic effect. An enhanced activation of p38 mitogen-activated protein kinase α (p38α MAPK) was identified. In line with this findings, p38 MAPK and, another important endoplasmic reticulum-stress-related kinase, c-Jun N-terminal kinase (JNK) were found to mediate the potentiated cytotoxic effect induced by the combination of MKC-8866 and nilotinib since the targeting of p38 MAPK with its specific inhibitor BIRB-796 or JNK with JNK-in-8 hindered the synergistic effect observed upon treatment with nilotinib and MKC-8866. In conclusion, the identified combined action of nilotinib and MKC-8866 might represent a successful therapeutic strategy in high-risk Ph+ ALL.
BCR-ABL1 inhibitor nilotinib and inositol-requiring enzyme 1 alpha inhibitor MKC-8866 are synergistic in Philadelphia-positive acute lymphoblastic leukemia and the activation of p38 mitogen-activated protein kinase/c-Jun N-terminal kinase pathways is the main mechanism at the basis of the synergism observed.
Accurate enumeration of circulating endothelial progenitor cells (CEP) is essential for their potential application as biomarkers of angiogenesis. In this study different stem cell markers (CD34, ...CD133) and endothelial cell antigens (KDR/VEGFR-2, CD31) in different flow cytometric protocols were assessed for the purpose of CEP quantification.
Blood samples from 19 healthy volunteers and 16 patients with different cancer types were analyzed by means of flow cytometry. Mononuclear cell gating was compared to additional gating on CD45-negative cells. CD34+/KDR+ and CD31+/CD133+ cells were analyzed in a direct immunolabeling approach. CEP were measured at different time points in individual patients and after storage of blood samples for 24
h and 48
h, respectively.
In contrast to previous studies, measurement of CD34+/KDR+ cells was an unreliable method for CEP enumeration, regardless of the applied gating strategy. However, detection of CD31+/CD133+ cells in a combined mononuclear cell and CD45-negative gating approach provided a reproducible method for CEP quantification. In individual blood donors, the CEP numbers were stable over time. For the first time, it was demonstrated that CEP are unstable in extracorporeal blood samples.
In this study, a reproducible protocol for CEP quantification was established. This protocol should facilitate future studies with the goal to further define the role of CEP as angiogenic biomarkers.
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