scholarly journals Leveraging Pathway-Interference to Overcome Drug-Resistance in Acute Lymphoblastic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 616-616
Author(s):  
Lai N. Chan ◽  
Mark A. Murakami ◽  
Christian Hurtz ◽  
Kohei Kume ◽  
Jaewoong Lee ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Signaling Pathways ◽  
Malignant Transformation ◽  
Small Molecule ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Oncogenic Pathways ◽  
Strong Negative Selection ◽  
Cancer Types ◽  
Oncogenic Driver

Abstract Background: The concept of multi-step carcinogenesis (Fearon and Vogelstein 1990) suggests that acquisition of mutations in addition to an existing set of mutations invariably accelerates tumor-progression. In colorectal cancer and many other cancer types, activation of multiple distinct oncogenic pathways is required for the development of invasive cancer. Here, we examined this paradigm for genetic lesions in B-ALL and 13 other cancer types. Bioinformatic approaches: To broadly study how oncogenic drivers across multiple signaling pathways interact, we developed a bioinformatic platform to map interactions between genetic lesions that cause oncogenic activation of eight oncogenic pathways, including PI3K, STAT5, NF-κB, Hippo, Notch, WNT, RAS-ERK and TGFβ-Smad pathways. Plotting of interaction scores between 56 pathway pairs in a matrix for 14 cancer types revealed that 12 of the 14 cancer types showed a pattern of globally synergistic pathway interactions, consistent with the Fearon and Vogelstein model of cooperation of multiple pathways to drive malignant transformation. Strikingly, B-ALL and gliomas showed the opposite behavior with largely antagonistic pathway interactions. Results. Unlike the vast majority of cancer types, B-ALL and gliomas are driven by one principal oncogenic pathway at a time. While the reasons for negative pathway interactions in glioma are unknown, we focused on functional analyses on pathway interference patterns in 1,148 cases of B-ALL. Genetic lesions leading to STAT5- or ERK-pathway activation are frequently found in B-ALL. Interestingly, activating lesions of both pathways co-occurred in only 3% of the cases studied, suggesting that co-activation of STAT5- and ERK-occurs much less frequently than expected by chance (odds ratio 0.13, P=2e-16, Figure, left). Unbiased interaction mapping analyses of mutational co-occurrence indicated strong negative selection for dual activation of both STAT5- and ERK-pathways (Figure, middle). Importantly this inter-pathway aversion is much stronger than intra-pathway effects, reflecting incompatibility rather than redundancy. Even in rare cases of co-occurrence in the same sample, single-cell mutation and phosphoprotein analyses revealed that STAT5- and ERK-activating mutations were mutually exclusive and reflected two competing clones. STAT5- and ERK-pathways engage conflicting transcriptional and biochemical programs, resulting in "friction", when both pathways are concurrently activated. In agreement with pathway interference, we demonstrated that Cre-mediated deletion of divergent pathway components - Erk2 fl/fl in a STAT5-driven model of B-ALL and Stat5 fl/fl in an ERK-driven B-ALL model - dramatically accelerated initiation of fatal leukemia in vivo. While Cre-mediated deletion of divergent pathway components precipitated leukemia-initiation, these findings suggest that reactivation of divergent signaling pathways represents a powerful barrier against malignant transformation. Interestingly, our preclinical studies suggested that pharmacological reactivation of divergent (suppressed) pathways can be leveraged for therapeutic benefit: The DUSP6 small molecule inhibitor BCI-215 functions as powerful activator of ERK and suppresses STAT5-phosphorylation, i.e. the principal pathway in STAT5-driven B-ALL (Figure, right). Likewise, DPH, a small molecule STAT5-agonist interferes with ERK-phosphorylation, the principal oncogenic driver in RAS-pathway B-ALL (Figure, right). Both BCI-215 and DPH significantly prolonged overall survival of NSG mice transplanted with refractory STAT5- and ERK-driven B-ALL PDX, respectively. Conclusions: We propose that a diverse spectrum of signaling input reflects interactions of normal cells with their environment, while convergence on one centralized pathway is a hallmark of cancer. Tracking early stages of leukemia-initiation, we identified convergence on one principal oncogenic driver and inactivation of diverging pathways as an early critical step. Pharmacological reactivation of divergent signaling pathways to subvert transformation was achievable by STAT5- and ERK-agonists. Proof-of-concept studies in patient-derived B-ALL cells revealed that pharmacological reactivation of suppressed divergent circuits can be leveraged as a previously unrecognized strategy to overcome drug-resistance. Figure 1 Figure 1. Disclosures Izraeli: Roche: Consultancy, Speakers Bureau; Bayer: Speakers Bureau; sightDx: Consultancy. Weinstock: ASELL: Consultancy; SecuraBio: Consultancy; Bantam: Consultancy; AstraZeneca: Consultancy; Abcuro: Research Funding; Verastem: Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Travera: Other: Founder/Equity; Ajax: Other: Founder/Equity.

scholarly journals SMYD3: An Oncogenic Driver Targeting Epigenetic Regulation and Signaling Pathways

Cancers ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 142 ◽  
Author(s):  
Cinzia Bottino ◽  
Alessia Peserico ◽  
Cristiano Simone ◽  
Giuseppina Caretti
Keyword(s):  
Signaling Pathways ◽  
Cancer Progression ◽  
Transcriptional Response ◽  
Migration And Invasion ◽  
Transcriptional Reprogramming ◽  
Oncogenic Pathways ◽  
Tumor Transformation ◽  
Cancer Types ◽  
Oncogenic Driver

SMYD3 is a member of the SMYD lysine methylase family and plays an important role in the methylation of various histone and non-histone targets. Aberrant SMYD3 expression contributes to carcinogenesis and SMYD3 upregulation was proposed as a prognostic marker in various solid cancers. Here we summarize SMYD3-mediated regulatory mechanisms, which are implicated in the pathophysiology of cancer, as drivers of distinct oncogenic pathways. We describe SMYD3-dependent mechanisms affecting cancer progression, highlighting SMYD3 interplay with proteins and RNAs involved in the regulation of cancer cell proliferation, migration and invasion. We also address the effectiveness and mechanisms of action for the currently available SMYD3 inhibitors. The findings analyzed herein demonstrate that a complex network of SMYD3-mediated cytoplasmic and nuclear interactions promote oncogenesis across different cancer types. These evidences depict SMYD3 as a modulator of the transcriptional response and of key signaling pathways, orchestrating multiple oncogenic inputs and ultimately, promoting transcriptional reprogramming and tumor transformation. Further insights into the oncogenic role of SMYD3 and its targeting of different synergistic oncogenic signals may be beneficial for effective cancer treatment.

scholarly journals Preclinical Activity of the MPS1 Inhibitor S81694 in Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2631-2631
Author(s):  
Anna Kaci ◽  
Emilie Adiceam ◽  
Melanie Dupont ◽  
Marine Garrido ◽  
Jeannig Berrou ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Solid Tumors ◽  
Cell Lines ◽  
Small Molecule ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Jurkat Cells ◽  
Cell Cycle Distribution ◽  
Monopolar Spindle

Introduction: The dual-specificity protein kinase, monopolar spindle 1 (Mps1) is one the main kinases of the spindle assembly checkpoint (SAC) critical for accurate segregation of sister chromatids during mitosis. A hallmark of cancer cells is chromosomal instability caused by deregulated cell cycle checkpoints and SAC dysfunction. Mps1 is known to be overexpressed in several solid tumors including triple negative breast cancer. Thus, Mps1 seems to be a promising target and small molecules targeting Mps1 entered clinical trials in solid tumors. ALL originates from malignant transformation of B-and T-lineage lymphoid precursors with a variety of genetic aberrations including chromosome translocations, mutations, and aneuploidies in genes responsible for cell cycle regulation and lymphoid cell development. While outcome is excellent for pediatric patients and younger adults, relapsed and refractory disease still remain a clinical challenge for elder patients. Here, we demonstrate for the first time preclinical efficacy of the small molecule Mps1 inhibitor (Mps1i) S81694 in T- and B- ALL cells including BCR-ABL1+-driven B-ALL. Materials and Methods: Expression of Mps1 was determined by RT-qPCR and WB in JURKAT, RS4-11 and BCR-ABL1+ cells (BV-173 and TOM-1). A small molecule Mps1i (S81694) was tested alone (0 to 1000nM) or in combination with imatinib, dasatinib, nilotinib and ponatinib in BCR-ABL1+ ALL cell lines. Cell viability and IC50 was assessed by MTS assays after exposure to Mps1i for 72h. In combination experiments, compounds were added simultaneously and relative cell numbers were determined at 72h with MTS assays and combination index (CI) values were calculated according to the Bliss model. Induction of apoptosis was evaluated by annexin-V exposure and PI incorporation at 72h with increasing doses of Mps1i. Cell-cycle distribution was determined by cytofluorometric analysis detecting nuclear propidium iodide (PI) intercalation at 48h. Phosphorylation of Mps1 was detected in synchronized (by nocodazole and MG-132) cells by immunofluorescence using an anti phospho-Mps1 antibody detecting Thr33/Ser37 residues. Time-lapse microscopy was used in cell lines in presence or absence of S81694 to determine mitosis duration. Bone marrow (BM) nucleated patient cells were obtained after informed consent and incubated in methylcellulose with cytokines with or without Mps1i for 2 weeks to determine colony growth. Results: Expression of Mps1 could be detected by RT-qPCR and at the protein level by WB in all cell lines (Figure 1A and B ). IC50 after Mps1i exposure alone was 126nM in JURKAT cells, 51nM in RS4-11 cells, 75nM in BV-173 cells and 83nM in TOM-1. Significant apoptosis as detected by phosphatidylserine exposure and PI incorporation in all cell lines with BCR-ABL1+ cell lines BV-173 and TOM-1 cells being the most sensitive (80% and 60% apoptotic cells respectively)(Figure 1C). Upon Mps1i exposure we observed targeted inhibition of Mps1 phosphorylation at Thr33/Ser37 residues indicating the specific on target effect of S81694 by inhibiting Mps1 autophosphorylation (Figure 1D and E). Cell cycle profile was generally lost after treatment with S81694 in all cell lines indicating aberrant 2n/4n distribution due to SAC abrogation (Figure 1F). Furthermore, we demonstrated that S81694 exposure accelerated significantly mitosis in BV-173 cell line from 36 minutes to 19 minutes indicating effective inhibition of SAC function (Figure 1G). Interestingly, S81694 induced significant apoptosis (70%) in the imatinib resistant BV173 cell line bearing the E255K-BCR-ABL1-mutation. Combination of S81694 with TKI imatinib, dasatinib and nilotinib (but not ponatinib) was strongly synergistic in BCR-ABL1+ cells (Figure 1H). Finally, we observed inhibition of colony formation in a patient with BCR-ABL1+ B-ALL after exposure to 100nM and 250nM S81694 (reduction of 85% and 100% respectively)(Figure 1I). Conclusion: Mps1i S81694 yields significant preclinical activity in T-and B-cell ALL including BCR-ABL1+ models. Interestingly S81694 was efficacious in a TKI resistant cell line. Disclosures Kaci: Institut de Recherches Internationales Servier (IRIS): Employment. Garrido:Institut de Recherches Internationales Servier (IRIS): Employment. Burbridge:Institut de Recherches Internationales Servier (IRIS): Employment. Dombret:AGIOS: Honoraria; CELGENE: Consultancy, Honoraria; Institut de Recherches Internationales Servier (IRIS): Research Funding. Braun:Institut de Recherches Internationales Servier (IRIS): Research Funding.

Phase I Trial of Nanomolecular Liposomal Annamycin in Adult Patients with Refractory Acute Lymphoblastic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4098-4098
Author(s):  
Meir Wetzler ◽  
Eunice S. Wang ◽  
Robert Shepard ◽  
Deborah A. Thomas ◽  
Michael Andreeff ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Phase I ◽  
Research Funding ◽  
Cardiac Toxicity ◽  
Lymphoblastic Leukemia ◽  
Tumor Lysis Syndrome ◽  
Study Drug ◽  
Adult Patients ◽  
Grade 3 ◽  
Mdr 1

Abstract Abstract 4098 There continues to be no effective second line therapy for refractory acute lymphoblastic leukemia (ALL) in adult patients and the cure rate with current therapy has not significantly improved in decades. Annamycin is a highly lipophilic form of the anthracycline doxorubicin with the ability to bypass multi-drug resistance (MDR-1) mechanisms of cellular drug resistance. A novel nanomolecular liposomal entity of annamycin was recently specifically synthesized which overcomes MDR with little to no cardiac toxicity and improved anti-tumor activity when compared to the original annamycin formulation. We performed a phase I multi-center, open-label, study to determine the maximally tolerated dose (MTD) of nanomolecular liposomal annamycin in adult patients with refractory ALL. The secondary objective was to study the MDR-1 encoded (permeability glycoprotein) PgP-170 glycoprotein expression in correlation with CD34 expression and MDR-1 mRNA levels in refractory ALL patients prior to and after receiving liposomal annamycin treatment. Thirty patients were enrolled on the study. The MTD was determined to be 150 mg/m2/day for 3 days. Other than the tumor lysis syndrome, there was only 1 severe adverse event (SAE) definitely related to the study drug consisting of grade 3 mucositis. There were also 3 other SAEs of grade 3-4 mucositis probably related to the study drug which comprised the MTD determination. There was no reported cardiac toxicity in any patients. After determining the MTD, a 10-patient phase IIA was conducted. Eight of the patients completed 1 cycle of the 3 days of treatment at the MTD. Of these, 5 (62%) had an efficacy signal with complete clearing of circulating peripheral blasts. Three of these subjects also cleared bone marrow blasts with one subsequently proceeding onto successful stem cell transplantation. The other 2 developed tumor lysis syndrome and unfortunately expired prior to response assessment. Nanomolecular liposomal annamycin appears to be well-tolerated with no cardiotoxicity and evidence of clinical activity as a single agent in refractory adult ALL. Given that the likely mechanism of action is overcoming innate drug resistance via MDR, PgP-170 mRNA and protein expression will be presented. We are currently testing liposomal annamycin in a phase I study in children and young adults with refractory ALL or AML, and planning a phase 2 registration trial in adult ALL Disclosures: Wetzler: Callisto Reserach Funding: Research Funding. Off Label Use: Annamycin. Refractory relapse to ALL. Wang:Callisto : Research Funding. Shepard:Callisto: Research Funding. Thomas:Callisto: Research Funding. Andreeff:Callisto: Research Funding. Kantarjian:Callisto: Research Funding.

scholarly journals Functional Genomics and Computational Approaches Identify Novel Small Molecules Targeting Quiescent Leukemia Stem Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1391-1391
Author(s):  
Costakis Frangou ◽  
Jason Den Haese ◽  
Jordan Warunek ◽  
Scott Portwood ◽  
Norma J Nowak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Drug Resistance ◽  
Signaling Pathways ◽  
Research Funding ◽  
Cell Tracking ◽  
Leukemia Stem Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Nsg Mice

Abstract Chemotherapy or targeted cancer therapies have greatly improved the treatment outcome of patients with leukemia; however, many will ultimately die because of disease relapse and development of drug resistance. Leukemias are cancers of the blood cells that result from alteration of the normal physiological constraints that regulate hematopoietic stem cells (HSCs). General characteristics of leukemia stem cells (LSCs) such as self-renewal, self-protection and proliferative quiescence represent inherent mechanisms that at least partially explain drug resistance and recurrence in post-therapy leukemia patients. Acute myeloid leukemia (AML) is a heterogeneous disease, both biologically and clinically, in which a number of distinct genetic abnormalities have been described. Several recent studies suggest that this heterogeneity extends to LSCs and can vary between patient subgroups, and even within individual patients. Moreover, the complexity of AML is further complicated by the existence of functionally diverse leukemic and preleukemic clones. Accordingly, the hierarchical organization of AML suggests that this may be relevant to current therapies that primarily target proliferating progenitors/blast cells, which lack self-renewal capacity, and not LSCs. In the current study, we rationalized that understanding how LSCs differ from normal HSCs at the molecular level, is an essential first step towards developing novel targeted therapies and achieving permanent disease remission. Despite the identification of novel LSC-specific markers, there is considerable heterogeneity in expression of these markers amongst AML patients. However, in addition to marker-enrichment strategies, LSCs can be identified by virtue of their quiescent and slow-cycling properties. For example, label-retaining cells can be isolated and used in functional assays but significant technical limitations impede broad utility of this approach. To this end, we describe the development and use of novel multi-fluorescent protein markers and DNA bar codes integrated into the cellular genomes by lentivirus, as single-cell tracking devices for monitoring LSCs in vivo. We demonstrate how LSCs can transition between a "proliferation phase" and a "quiescence phase" in vivo. Furthermore, using high-throughput quantitative transcriptome sequencing (Q-RNA-Seq) and RNAi genetic perturbation's focusing on well-defined self-renewal signaling pathways, we develop a differential network-based model to identify LSC-specific genes and subsequently prioritize/rank candidates as potential drug targets. In the current study, we identify several molecular targets deregulated in quiescent versus proliferating LSCs and a mutual set of signaling pathways that facilitate leukemic transformation downstream of diverse initiating mutations/lesions. Remarkably, both quiescent and dividing LSCs but not HSCs, were 'addicted' to SSRP1 - an essential component of the ubiquitous FACT chromatin remodeling complex. Two orally available quinacrine-related DNA-intercalating compounds inhibiting function of FACT (CBL0100 and CBL0175, respectively) suppressed LSC proliferation in vitro and in vivo, as demonstrated by production of leukemic clonogenic cells (CFU) and long-term engraftment of immunodeficient NSG mice, by simultaneous inhibition of NF-kB (stimulated and basal forms) and activation of p53. Furthermore, in a secondary transplantation experiment, leukemic cells obtained from CBL0175 treated mice (primary) failed to engraft into secondary NSG mice in a serial transplantation model by selectively targeting the LSC compartment. Collectively, we present a novel network-based polypharmacology approach that provides unique opportunities to preferentially ablate LSCs (quiescent and dividing types), with potentially profound clinical implications. Disclosures Frangou: Cellecta: Employment. Portwood:ImmunoGen: Research Funding. Wang:ImmunoGen: Research Funding.

scholarly journals AVA4746, an Orally Available, Clinical Grade Antagonist of Integrin Alpha 4, Sensitizes Pre-B Cell Acute Lymphoblastic Leukemia to Chemotherapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2765-2765 ◽  
Author(s):  
Yongsheng Ruan ◽  
Eun Ji Gang ◽  
Hye-Na Kim ◽  
Chintan Parekh ◽  
Hisham Abdel-Azim ◽  
...  
Keyword(s):  
Overall Survival ◽  
Drug Resistance ◽  
Acute Lymphoblastic Leukemia ◽  
Metabolic Activity ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Drug Resistant ◽  
Support Research

Abstract Background. Even though remarkable progress has been made in the treatment of childhood acute lymphoblastic leukemia (ALL), salvage of relapse patients remains a challenge. The role of the bone marrow (BM) microenvironment is critical to protect leukemia cells from chemotherapy. The BM microenvironment promotes cell adhesion-mediated drug resistance (CAM-DR) in ALL.We and others have shown that the adhesion molecule integrin α4, referred to hereafter as α4, mediates drug resistance of B-ALL. In our previous studies, we showed that both α4 blockade by natalizumab and inhibition by the small molecule α4 antagonist TBC3486 can sensitize relapsed ALL cells to chemotherapy. However, no α4 targeting therapy is currently clinically available to treat leukemia. Here, we preclinically evaluate a novel non-peptidic small molecule antagonist, AVA4746, which has been safely used in clinical studies, as a potential new approach to combat drug resistant ALL. Method. Six refractory or relapsed primary pre-B ALL cases were used for in vitro studies. Viability was assessed by trypan blue counts or annexin V/7AAD flow cytometric analysis and metabolic activity was evaluated by Cytoscan WST-1 assay. For in vivo evaluation a NOD/SCID IL2Rγ-/- xenograft model of primary pre-B ALL (LAX7R) was used.AVA4746 (15mg/kg) was administered by oral gavage twice a day continuously for 14 days, and vincristine, dexamethasone, L-asparaginase (VDL) was given intraperitoneally (weekly) for 4 weeks. Overall survival was determined by Kaplan-Meier Survival analysis. Results. AVA4746 caused a significant decrease in mean fluorescence intensity (MFI) of α4 expression in six out of six ALL cases at doses of both 5μM and 25μM after 24 hours and 96 hours compared to DMSO control. Interestingly, decreased protein expression of α4 was also observed by Western Blot analysis all six ALL cases. We tested next in two cases (LAX53, ICN13), if AVA4746 de-adheres ALL cells from its counter receptor VCAM-1. The percentages of adherence after treatment with AVA4746 (25μM) were significantly lower than after DMSO treatment (10.3%±4.9% vs. 99.9%±7.6%, p= 0.00007 for LAX7R; 8.1%±1.0% vs. 100.1%±13.6%, p= 0.0003 for LAX53; 9.0%±1.6% vs. 100.0%±14.0%, p=0.0004 for ICN13). AVA4746 was not associated with apoptosis in vitro alone or in combination with chemotherapy (VDL). Metabolic activity as assessed by WST-1 assay was markedly decreased by AVA4746 in two of two ALL cases. AVA4746 also decreased ALL proliferation in two out of two ALL samples tested. In vivo, AVA4746 in combination with VDL chemotherapy treatment led to significant prolongation of overall survival (n=6) compared with the VDL only treated group (n=6) (MST= 78.5 days vs MST= 68 days; P<0.05). There was no significant difference in survival between the PBS control group (n=5) and the AVA4746 mono-treatment group (n=5) (MST=38days vs MST= 38days). Conclusion. We have identified α4 as a central adhesion molecule in CAM-DR of ALL and have shown that AVA-4746, an orally available and specific α4 antagonist, which has been safely used in clinical studies, downregulates α4 in primary ALL and functionally de-adheres them from VCAM-1. Critically, we demonstrated that inhibition of α4 in combination with standard chemotherapy can prolong the survival of NSG mice bearing pre-B ALL. These data support further study of inhibition of α4 using AVA4746 as a novel strategy to treat drug resistant B lineage ALL. Disclosures Bhojwani: Amgen: Other: Blinatumumab global pediatric advisory board 2015. Wayne:Spectrum Pharmaceuticals: Honoraria, Other: Travel Support, Research Funding; Kite Pharma: Honoraria, Other: Travel support, Research Funding; Pfizer: Consultancy, Honoraria, Other: Travel Support; Medimmune: Honoraria, Other: Travel Support, Research Funding; NIH: Patents & Royalties. Kim:Antisense Therapeutics Ltd: Patents & Royalties.

Overexpressed A20 in Refractory/Relapse B-ALL May Serve As a Potential Therapeutic Target

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4816-4816
Author(s):  
Yi Xu ◽  
Xiuli Wu ◽  
Fan Zhang ◽  
Sichu Liu ◽  
Shaohua Chen ◽  
...  
Keyword(s):  
Drug Resistance ◽  
B Cell ◽  
Natural Science ◽  
Research Funding ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Healthy Controls ◽  
Newly Diagnosed ◽  
Potential Therapeutic Target ◽  
Expression Levels

Abstract Abstract 4816 Treatment failure of precursor B-lymphoblastic leukemia (B-ALL), also known as B-cell acute lymphoblastic leukemia and B-cell acute lymphocytic leukemia, remains a formidable problem. The molecular abnormalities responsible for relapse or drug resistance are still not clear. NF-κB-dependent gene expression and apoptosis plays crucial roles in numerous cellular processes, and defects in their regulation may contribute to a variety of diseases like cancers. As a key regulatory component of the cytoplasmic signaling cascade that mediates NF-κB activation in response to DNA damage, the zinc finger-containing protein A20 (also known as TNF-α-induced protein 3, TNFAIP3) has been found to correlate with drug resistance of breast cancer, prostate cancer and acute myeloid leukemia. To investigate the function of A20 in the development of B-ALL, we analyzed the expression levels of A20 and NF-κB genes in peripheral blood mononuclear cells (PBMCs) from patients (aged 3–72 years, median 30.5) with newly diagnosed B-ALL, B-ALL in complete remission (CR), and refractory/relapse B-ALL. Twenty-nine healthy individuals (aged 20–57, median 28) served as controls. We found that the expression levels of A20 in newly diagnosed B-ALL patients (23.450±16.164) and refractory/relapse B-ALL patients (26.108±12.301) were significantly higher than those from healthy controls (9.865±5.370) (P<0.0001, P<0.0001), while the expression levels of A20 in B-ALL CR patients (2.826±2.415) were significantly decreased and was lower than those from healthy controls (P=0.008). The expression levels of NF-κB in newly diagnosed B-ALL patients (2.025±0.986) were significantly higher than healthy controls (0.337±0.311) (P<0.0001), while the expression levels of NF-κB in refractory/relapse B-ALL patients (0.668±0.707) and B-ALL CR patients (0.405±0.172) were similar to healthy group (P=0.057, P=0.559). The expression levels of A20 had a positive correlation with the expression levels of NF-κB in de novo B-ALL (rs=0.7473, P=0.021), however, the expression levels of A20 in refractory/relapse B-ALL and B-ALL CR group did not show significantly correlation with the expression levels of NF-κB. In conclusion, overexpression of A20 is a feature in de novo and refractory/relapse B-ALL, which may loss their negative regulating function for NF-κB. The expression level of A20 may correlate with the clinical stages of B-ALL, indicating A20 might be considered as a prognostic marker of B-ALL and a potential therapeutic target for refractory/relapse B-ALL. Disclosures: Xu: National Natural Science Foundation of China (No. 91129720): Research Funding. Wu:National Natural Science Foundation of China (No. 91129720): Research Funding. Zhang:National Natural Science Foundation of China (No. 91129720): Research Funding. Liu:National Natural Science Foundation of China (No. 91129720): Research Funding. Chen:National Natural Science Foundation of China (No. 91129720): Research Funding. Yang:National Natural Science Foundation of China (No. 91129720): Research Funding. Li:National Natural Science Foundation of China (No. 91129720): Research Funding. Li:National Natural Science Foundation of China (No. 91129720): Research Funding.

YB-1 Is Activated by IL-7 and Is Overexpressed in Pediatric Pre-B Acute Lymphoblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1468-1468
Author(s):  
Amina Kariminia ◽  
Sabine M Ivison ◽  
Vivian Leung ◽  
Sandra E. Dunn ◽  
Aru Narendran ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Drug Resistance ◽  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
B Cell Lymphoma ◽  
Fluorescent Intensity

Abstract Abstract 1468 Background: Increased YB-1 expression correlates with poor prognosis, drug resistance and metastasis in several different cancers including B cell lymphoma. Phosphorylation and nuclear localization of YB-1 in response to growth factors leads to increased survival through expression of proteins such as survivin and multidrug resistance protein 1. Until now, its role in leukemia has not been investigated. We hypothesized that YB-1 expression is aberrantly regulated in pediatric pre-B acute lymphoblastic leukemia (pre-B ALL), and that YB-1 may be activated downstream of IL-7. This cytokine facilitates the differentiation and survival of pre-B cells and has been implicated in the drug resistance of pre-B ALL. Methods: YB-1 and IL-7Ra protein expression was investigated by flow cytometry in normal pre-B cells (CD19+CD10+CD20−), and mature B cells (CD19+CD10−CD20+) as well as diagnostic and relapsed pre-B ALL (CD19+CD10+/−). Cell surface and cytoplasmic expression was quantified by mean fluorescent intensity (MFI). Bone marrow from healthy donors was used as a source of normal pre-B cells, while mature B cells were derived from PBMCs; leukemic cells at presentation and relapse were obtained following local IRB approval and informed consent. Activation of YB-1 downstream of IL-7 stimulation (25 ng/ml) was examined in pre-B ALL cell lines or NSG (NOD scid gamma) mice-expanded pre-B ALL by Western blotting using anti-phosphoYB-1(S012). Pre-B ALL cell lines used in these experiments were 697, 380, RCH and RS-4;11. Signaling pathways were investigated by pre-treatment of cells with pharmacological inhibitors followed by Western analyses. For the transient overexpression of YB-1, pEGFP or a pEGFP/YB-1 fusion protein was electroporated into freshly isolated mature B cells (which have a low basal expression of YB-1) and YB-1 and IL-7Ra expression was assessed by flow cytometry after 24 h. Results: While intracellular YB-1 expression was significantly higher in leukemia samples at presentation compared to normal pre-B cells, the highest YB-1 levels were found in relapsed pre-B ALL (see figure, part A). All examined pre-B ALL cell lines had levels comparable to those of the relapse samples. Similarly, surface IL-7Ra (CD127) levels (MFI medians; upper-lower range) were increased in onset (221; 150–286), and relapsed (1840; 651–2030) ALL compared to normal pre-B cells (528; 333–2673). (normal pre-B vs. leukemia at presentation, p<0.001, Mann-Whitney). Overexpression of YB-1-GFP in normal mature B cells resulted in increased expression of IL-7Ra (see figure, part B), suggesting an link between the YB-1 and IL-7 signaling pathways. Activated YB-1 is phosphorylated on S102 and relocated to the nucleus. Addition of IL-7 to pre-B ALL cell lines led to phosphorylation of YB-1 within 30 min. Similar results were shown for patient-derived, NSG mice-expanded pre-B ALL samples. Intracellular immunostaining using Imagestream technology (Amnis) showed that IL-7 treatment of pre-B ALL cell lines increased nuclear YB-1 levels 4-fold. As PI3K and MEK1 are involved in signaling downstream of IL-7, we investigated their role in YB-1 signaling in both pre-B ALL cell lines and NSG-mouse expanded pre-B ALL using pharmacological inhibitors. Western analyses showed that inhibition of PI3K using LY294002 did not prevent IL-7-mediated phosphorylation of YB-1 but the MEK1 inhibitor U0126 did, indicating the involvement of MAPK (see figure, part C). Conclusion: We show that YB-1, which is highly expressed in pediatric pre-B ALL compared to normal pre-B cells, is expressed at even higher levels after relapse. We demonstrate a link between the YB-1 and IL-7 signaling pathways which could offer a novel target for the treatment of refractory leukemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Signaling Input from Divergent Pathways Subverts Malignant B-Cell Transformation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3944-3944
Author(s):  
Lai N Chan ◽  
Mark A. Murakami ◽  
Rebecca Caesar ◽  
Christian Hurtz ◽  
Kohei Kume ◽  
...  
Keyword(s):  
B Cell ◽  
Signaling Pathways ◽  
Malignant Transformation ◽  
Cell Transformation ◽  
Cell Receptor ◽  
Current Treatment ◽  
Advisory Committees ◽  
Surface Receptors ◽  
Oncogenic Driver ◽  
Principal Pathway

Background and significance: Normal B-cells constantly exchange information with their environment and depend on external cues for proliferation and survival that engage multiple divergent pathways (e.g. cytokine receptors; B-cell receptor, BCR). The dependency of normal B-cells on signaling input from a diverse repertoire of surface receptors is in contrast to transforming oncogenes that engage one single pathway. Beyond the established concept that diverse signal input from multiple cell surface receptors becomes dispensable in transformed cells, we here provide evidence that inactivation of divergent pathways that are not aligned with the principal oncogenic driver represents a critical step during malignant transformation. Tracking early stages of leukemia-initiation, we identified convergence on one principal oncogenic driver and inactivation of diverging pathways as critical events during B-cell transformation. Our results support a scenario in which reactivation of divergent and potentially conflicting signaling pathways represents a powerful barrier to malignant transformation. Here we studied the interaction of STAT5- and ERK-signaling pathways during normal B-cell development and malignant B-cell transformation, and found that convergence on one principal oncogenic driver represents a critical event during B cell transformation and a previously unrecognized vulnerability. Results: Our analysis of 987 patient-derived B-cell acute lymphoblastic leukemia leukemia (B-ALL) samples revealed that individual mutations did not promote leukemogenesis unless they converged on one single oncogenic pathway. Mutations that were not aligned with the central oncogenic driver would activate divergent pathways and subvert malignant transformation. Oncogenic lesions in B-ALL frequently mimic survival and proliferation signals downstream of cytokine receptors (STAT5) or the B cell receptor (ERK). STAT5- (286 cases) and ERK- (386 cases) activating lesions were frequently found but rarely co-occurred in the same sample (35 cases; P=2.5E-16). Single-cell mutation and phosphoprotein analyses revealed that even in these rare cases, oncogenic STAT5- or ERK-activation were mutually exclusive and segregated to competing clones. STAT5 and ERK engaged opposing biochemical and transcriptional programs orchestrated by MYC and BCL6, respectively. Genetic reactivation of the divergent (suppressed) pathway came at the expense of the principal oncogenic driver and reversed malignant transformation. Conversely, Cre-mediated deletion of divergent pathway components triggered leukemia-initiation and accelerated development of fatal disease. Thus, persistence of divergent signaling pathways represents a powerful barrier to malignant transformation and convergence on one principal driver defines a key event during leukemia-initiation. Proof-of-concept studies in patient-derived B-ALL cells revealed that small molecule agonists of STAT5 or ERK to reactivate the suppressed divergent circuits subvert oncogenic signaling and strongly synergized with direct inhibition of the principal oncogenic driver. Hence, pharmacological reactivation of divergent pathways can be leveraged as a previously unrecognized strategy to deepen treatment responses and to overcome drug-resistance. Current treatment approaches for drug-resistant cancer are focused on drug-combinations to suppress the central oncogenic driver and multiple alternative pathways. Here, we introduce a concept based on inhibition of the principal driver combined with pharmacological reactivation of divergent pathways Conclusions: These results provide evidence that inactivation of divergent pathways that are not aligned with the principal oncogenic driver represents a critical step during malignant transformation. Unlike B-ALL, where reactivation of a divergent pathway suppresses the principal pathway and compounds toxicity, activation of an alternative pathway in solid tumors represents a route for survival and drug resistance. While current treatment approaches for drug-resistant cancer are focused on drug-combinations to inhibit multiple pathways, we introduce a scenario that is based on inhibition of the principal pathway combined with reactivation of divergent pathways. Figure Disclosures Wiita: UCSF: Patents & Royalties; Indapta Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Protocol Intelligence: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Izraeli:sightdx: Consultancy; novartis: Honoraria; prime oncology: Speakers Bureau. Weinstock:Celgene: Research Funding; Verastem Oncology: Research Funding.

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