Activation of a Novel Proteasomal Independent Bcr/Abl Degradation Pathway by WP1130 Induces Apoptosis in CML Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2862-2862
Author(s):  
Geoffrey A. Bartholomeusz ◽  
Nichalos Donato ◽  
Zeev Estrov ◽  
Waldemar Priebe ◽  
Moshe Talpaz
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Tyrosine Phosphatase ◽  
Point Mutations ◽  
Effective Therapy ◽  
Myelogenous Leukemia ◽  
Small Molecular Weight ◽  
Hematopoietic Stem ◽  
Clinical Resistance

Abstract Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder of hematopoietic stem cells caused by 9:22 reciprocal chromosomal translocation resulting in expression of a highly stable, constitutively active tyrosine kinase, Bcr/Abl. Inhibition of Bcr/Abl with imatinib mesylate, a potent Abl-specific tyrosine kinase inhibitor, is a highly effective therapy for this disease. However, clinical resistance occurs particularly in the later stages of the disease due mainly to the occurrence of point mutations in the Abl kinase domain and continual Bcr/Abl signaling. Alternative and supplemental therapies are still needed and of great clinical interest. Screening a library of small molecular weight compounds for their ability to induce the degradation of critical transcription factors and tyrosine kinases led to the design and synthesis of WP1130, a compound that reduces Jak2 and Bcr/Abl protein stability. Treatment with WP1130 caused the rapid destruction of Bcr/Abl in human CML cell lines (K562, BV173) as well as in clinical CML specimens obtained from patients. This treatment also resulted in dephosphorylation of pSTAT5, pHck and pCrkL, three known targets of Bcr/Abl. Loss of Bcr/Abl protein following WP1130 treatment was similar to that caused by Bcr/Abl silencing (siRNA), and both treatments lead to the induction of apoptosis. Treatment with WP1130 caused rapid degradation of both wild-type and mutant (T315I) Bcr/Abl protein in BaF3 cell expressing these proteins and in a CML cell line expressing wildtype (BV173) and T315I mutant BCR-ABL (BV-173R). Treatment with WP1130 also strongly inhibited colony formation in soft agar of human CML cells from imatinib resistant patients expressing the T315I mutation. The degradation of Bcr/Abl in cells treated with WP1130 was rapid, (1h) and independent of the proteasomal system and HSP90, a chaperone associated with Bcr/Abl stability. Interestingly, WP1130-induced Bcr/Abl degradation was blocked in the presence of vanadate, a tyrosine phosphatase inhibitor. Together, these observations suggest that WP1130-induced Bcr/Abl degradation is mediated by a unique, proteasomal independent pathway in CML cells. Further development of WP1130 is underway with the goal of developing an effective therapy for treating CML by targeting Bcr/Abl protein expressed in early CML progenitors or stem cell populations.

Efficacy of STI571, an Abl tyrosine kinase inhibitor, in conjunction with other antileukemic agents against Bcr-Abl–positive cells

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3195-3199 ◽  
Author(s):  
J. Tyler Thiesing ◽  
Sayuri Ohno-Jones ◽  
Kathryn S. Kolibaba ◽  
Brian J. Druker
Keyword(s):  
Clinical Trials ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Kinase Inhibitor ◽  
Single Agent ◽  
Myelogenous Leukemia ◽  
Hematopoietic Stem ◽  
Abl Tyrosine Kinase ◽  
Abl Tyrosine Kinase Inhibitor

Abstract Chronic myelogenous leukemia (CML), a malignancy of a hematopoietic stem cell, is caused by the Bcr-Abl tyrosine kinase. STI571(formerly CGP 57148B), an Abl tyrosine kinase inhibitor, has specific in vitro antileukemic activity against Bcr-Abl–positive cells and is currently in Phase II clinical trials. As it is likely that resistance to a single agent would be observed, combinations of STI571 with other antileukemic agents have been evaluated for activity against Bcr-Abl–positive cell lines and in colony-forming assays in vitro. The specific antileukemic agents tested included several agents currently used for the treatment of CML: interferon-alpha (IFN), hydroxyurea (HU), daunorubicin (DNR), and cytosine arabinoside (Ara-C). In proliferation assays that use Bcr-Abl–expressing cells lines, the combination of STI571 with IFN, DNR, and Ara-C showed additive or synergistic effects, whereas the combination of STI571 and HU demonstrated antagonistic effects. However, in colony-forming assays that use CML patient samples, all combinations showed increased antiproliferative effects as compared with STI571 alone. These data indicate that combinations of STI571 with IFN, DNR, or Ara-C may be more useful than STI571 alone in the treatment of CML and suggest consideration of clinical trials of these combinations.

scholarly journals The quiescent fraction of chronic myeloid leukemic stem cells depends on BMPR1B, Stat3 and BMP4-niche signals to persist in patients in remission

Haematologica ◽  
2020 ◽  
Vol 106 (1) ◽  
pp. 111-122 ◽  
Author(s):  
Sandrine Jeanpierre ◽  
Kawtar Arizkane ◽  
Supat Thongjuea ◽  
Elodie Grockowiak ◽  
Kevin Geistlich ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Tyrosine Kinase ◽  
Stromal Cells ◽  
Kinase Inhibitor ◽  
Bmp Signaling ◽  
Myelogenous Leukemia ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem

Chronic myelogenous leukemia arises from the transformation of hematopoietic stem cells by the BCR-ABL oncogene. Though transformed cells are predominantly BCR-ABL-dependent and sensitive to tyrosine kinase inhibitor treatment, some BMPR1B+ leukemic stem cells are treatment-insensitive and rely, among others, on the bone morphogenetic protein (BMP) pathway for their survival via a BMP4 autocrine loop. Here, we further studied the involvement of BMP signaling in favoring residual leukemic stem cell persistence in the bone marrow of patients having achieved remission under treatment. We demonstrate by single-cell RNA-Seq analysis that a sub-fraction of surviving BMPR1B+ leukemic stem cells are co-enriched in BMP signaling, quiescence and stem cell signatures, without modulation of the canonical BMP target genes, but enrichment in actors of the Jak2/Stat3 signaling pathway. Indeed, based on a new model of persisting CD34+CD38- leukemic stem cells, we show that BMPR1B+ cells display co-activated Smad1/5/8 and Stat3 pathways. Interestingly, we reveal that only the BMPR1B+ cells adhering to stromal cells display a quiescent status. Surprisingly, this quiescence is induced by treatment, while non-adherent BMPR1B+ cells treated with tyrosine kinase inhibitors continued to proliferate. The subsequent targeting of BMPR1B and Jak2 pathways decreased quiescent leukemic stem cells by promoting their cell cycle re-entry and differentiation. Moreover, while Jak2-inhibitors alone increased BMP4 production by mesenchymal cells, the addition of the newly described BMPR1B inhibitor (E6201) impaired BMP4-mediated production by stromal cells. Altogether, our data demonstrate that targeting both BMPR1B and Jak2/Stat3 efficiently impacts persisting and dormant leukemic stem cells hidden in their bone marrow microenvironment.

Role for tyrosine kinases in carbachol-regulated Ca entry into colonic epithelial cells

1995 ◽  
Vol 268 (1) ◽  
pp. C154-C161 ◽  
Author(s):  
G. Bischof ◽  
B. Illek ◽  
W. W. Reenstra ◽  
T. E. Machen
Keyword(s):  
Epithelial Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Divalent Cations ◽  
Tyrosine Phosphatase ◽  
Inhibitory Effect ◽  
Colonic Epithelial Cells

We studied a possible role of tyrosine kinases in the regulation of Ca entry into colonic epithelial cells HT-29/B6 using digital image processing of fura 2 fluorescence. Both carbachol and thapsigargin increased Ca entry to a similar extent and Ca influx was reduced by the tyrosine kinase inhibitor genistein (50 microM). Further experiments were performed in solutions containing 95 mM K to depolarize the membrane potential, and the effects of different inhibitors on influx of Ca, Mn, and Ba were compared. Genistein, but not the inactive analogue daidzein nor the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2- methylpiperazine, decreased entry of all three divalent cations by 47-59%. In high-K solutions, carbachol or thapsigargin both caused intracellular Ca to increase to a plateau of 223 +/- 19 nM. This plateau was reduced by the tyrosine kinase inhibitors genistein (to 95 +/- 8 nM), lavendustin A (to 155 +/- 17 nM), and methyl-2,5-dihydroxycinnamate (to 39 +/- 3 nM). Orthovanadate, a protein tyrosine phosphatase inhibitor, prevented the inhibitory effect of genistein. Ca pumping was unaffected by genistein. Carbachol increased tyrosine phosphorylation (immunoblots with anti-phosphotyrosine antibodies) of 110-, 75-, and 70-kDa proteins, and this phosphorylation was inhibited by genistein. We conclude that carbachol and thapsigargin increase Ca entry, and tyrosine phosphorylation of some key proteins may be important for regulating this pathway.

Overcoming Resistance in Chronic Myelogenous Leukemia

2013 ◽  
pp. 306-312
Author(s):  
Michael J. Mauro
Keyword(s):  
Chronic Myelogenous Leukemia ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Point Mutations ◽  
Kinase Domain ◽  
Drug Binding ◽  
Myelogenous Leukemia ◽  
Novel Therapy ◽  
Abl Kinase ◽  
Clinical Resistance

Resistance in chronic myelogenous leukemia is an issue that has developed in parallel to the availability of rationally designed small molecule tyrosine kinase inhibitors to treat the disease. A significant fraction of patients with clinical resistance are recognized to harbor point mutations/substitutions in the Abl kinase domain, which limit or preclude drug binding and activity. Recent data suggest that compound mutations may develop as well. Proper identification of clinical resistance and prudent screening for all causes of resistance, ranging from adherence to therapy to Abl kinase mutations, is crucial to success with kinase inhibitor therapy. There is currently an array of Abl kinase inhibitors with unique toxicity and activity profiles available, allowing for individualizing therapy beginning with initial choice at diagnosis and as well informed choice of subsequent therapy in the face of toxicity or resistance, with or without Abl kinase domain mutations. Recent studies continue to highlight the merits of increasingly aggressive initial therapy to subvert resistance and importance of early response to identify need for change in therapy. Proper knowledge and navigation amongst novel therapy options and consideration of drug toxicities, individual patient characteristics, disease response, and vigilance for development of resistance are necessary elements of optimized care for the patient with chronic myelogenous leukemia.

Generation of Specific Resistance Profiles in FLT3-ITD and FIP1L1-PDGFRalpha towards Specifically Acting Tyrosine Kinase Inhibitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1376-1376
Author(s):  
Nikolas von Bubnoff ◽  
Silvia Thoene ◽  
Sivahari P. Gorantla ◽  
Jana Saenger ◽  
Christian Peschel ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Point Mutations ◽  
Kinase Domain ◽  
Myelogenous Leukemia ◽  
Resistance Mutations ◽  
Abl Kinase ◽  
Mechanism Of Resistance

Abstract BCR-ABL kinase domain mutations constitute the major mechanism of resistance in patients with chronic myelogenous leukemia treated with the ABL kinase inhibitor imatinib. Mutations causing resistance to therapeutic kinase inhibition were also identified in other target kinases in various malignant diseases, such as FLT3-ITD in acute myelogenous leukemia, cKit in gastrointestinal stromal tumors, EGFR in patients with lung cancer, and FIP1L1-PDGFRalpha in hypereosinophilic syndrome. Thus, mutations in kinase domains seem to be a general mechanism of resistance to therapeutically applicated tyrosine kinase inhibitors. We recently developed a cell-based screening strategy that allows one to predict the pattern and relative abundance of BCR-ABL resistance mutations emerging in the presence of imatinib, and the novel ABL kinase inhibitor AMN107 (nilotinib). We therefore intended to determine, if this method would also allow the generation of resistant cell clones with other oncogeneic tyrosine kinases as targets in the presence of specifically acting kinase inhibitors. When FLT3-ITD and su5614 were used as drug/target combination in our cell-based method, the frequency of resistant clones in the presence of su5614 at 10 times the IC50 was 0.17 per million cells. In 40 per cent of resistant clones, point mutations were detected leading to amino acid exchanges within the FLT3-ITD split kinase domain. The yield of resistant clones was increased by the factor of 14 to 2.37 per million cells by adding ethyl-nitrosourea (ENU), a potent inducer of point mutations. Also, the proportion of mutant clones increased from 40 to 74 per cent. In 83 mutant clones that were examined so far, we detected eight exchanges affecting kinase domain two (TK2) of the split kinase domain within or shortly behind the FLT3-ITD activation loop (A-loop). We did not detect exchanges affecting TK1. We next examined whether resistant clones would also come up with FIP1L1-PDGFRalpha-transformed cells in the presence of imatinib. Again, the yield of resistant clones increased when cells were pretreated with ENU, and a proportion of resistant clones contained mutations in the FIP1L1-PDGFRalpha kinase domain, affecting the nucleotide-binding loop (P-loop) and A-loop. We conclude that cell-based resistance screening is a simple and powerful tool that allows prediction of resistance mutations towards kinase inhibitors in various relevant oncogeneic kinases.

Phosphoproteomic Profiling of the Signaling Output of FLT3-ITD and Its AC220-Resistant Mutants Reveals Profound Signaling Differences and Differential Responsiveness to Inhibition of Downstream Kinases

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2453-2453
Author(s):  
Carmen Doebele ◽  
Johannes Kovar ◽  
Diego Yepes ◽  
Silvia Münch ◽  
Frank Schnütgen ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Conflicts Of Interest ◽  
Remission Rate ◽  
Point Mutations ◽  
Tyrosine Kinase Domain ◽  
Hematopoietic Stem ◽  
Differential Activation ◽  
Flt3 Inhibitor ◽  
Clinical Resistance ◽  
Resistant Mutants

Abstract Acute myeloid leukemia (AML) is a genetically heterogeneous disease where multiple mutations coincide in hematopoietic stem and progenitor cells leading to malignant transformation. One important class of mutations alters the function of signaling intermediates such as Fms-like tyrosine kinase 3 (FLT3), thereby helping AML cells to overcome the physiological communication with their microenvironment. Activating mutations in FLT3 are found in approximately 30% of adult AML cases. Particularly common are internal tandem duplications (ITD) in the juxtamembrane domain of FLT3, which are associated with poor clinical outcome. Recently, a phase II study of the second-generation FLT3 inhibitor AC220 (quizartinib) showed a complete remission rate of 44% to 54% in relapsed/chemotherapy refractory AML. However, secondary point mutations in the FLT3 tyrosine kinase domain have been reported as common causes of acquired clinical resistance to the FLT3 inhibitor AC220. We used quantitative mass-spectrometry-based phosphoproteomics to elucidate and compare the signaling out-put of FLT3-ITD and its AC220-resistant mutants harbouring either the F691L 'gatekeeper' substitution or the D835V activation loop mutant in AML cell models. Our comprehensive signaling analyses profiled thousands of phosphorylation events in a site-specific manner and revealed marked differences in the signaling profiles of the FLT3 mutant variants. In general, we found differential activation of signal transducer and activator of transcription 5 (STAT5) and mitogen-activated protein (MAP) kinase signaling when comparing FLT3-ITD and the AC220-resistant mutants. Interestingly, some cytosolic tyrosine kinases showed differential activation patterns. For instance, spleen tyrosine kinase (SYK) signaling was significantly enhanced downstream of FLT3-ITD-F691L and cells harbouring this mutant showed increased responsiveness to compounds targeting SYK. Our resource study shows, how point mutations conferring resistance to AC220 impact the signaling output of FLT3-ITD and uncovers pathways whose inhibition might be useful to disrupt oncogenic signaling elicited by FLT3-ITD-F691L and -D835V mutants. Disclosures No relevant conflicts of interest to declare.

scholarly journals Tyrosine Kinase Inhibitor Use in Pediatric Philadelphia Chromosome–Positive Acute Lymphoblastic Anemia

Hematology ◽  
2011 ◽  
Vol 2011 (1) ◽  
pp. 361-365 ◽  
Author(s):  
Stephen P. Hunger
Keyword(s):  
Tyrosine Kinase ◽  
Second Generation ◽  
Kinase Inhibitor ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Myelogenous Leukemia ◽  
Kinase Signaling ◽  
New Paradigm ◽  
Hematopoietic Stem ◽  
Philadelphia Chromosome Positive

Abstract Until recently, pediatric Philadelphia chromosome–positive (Ph+) acute lymphoblastic leukemia (ALL) was associated with an extremely poor outcome when treated with chemotherapy alone, and only modest survival benefits were obtained with the widespread use of hematopoietic stem cell transplantation (HSCT). The development of first-generation (imatinib) and second-generation (dasatinib and nilotinib) tyrosine kinase inhibitors (TKIs) that target the BCR-ABL1 fusion protein produced by the Ph chromosome revolutionized the treatment of chronic myelogenous leukemia (CML). The Children's Oncology Group (COG) AALL0031 trial showed that the addition of imatinib to intensive chemotherapy did not cause increased toxicity and resulted in 3-year event-free survival rates that were more than double those of historical control data from the pre-imatinib era. These findings create a new paradigm for integrating molecularly targeted agents with conventional chemotherapy and call for a reassessment of the routine use of HSCT for children and adolescents with Ph+ ALL. Second-generation TKIs have theoretical advantages over imatinib, and are now being tested in Ph+ ALL. The focus of contemporary trials is to define the optimal use of chemotherapy, HSCT, and TKI in Ph+ ALL. In the coming years, it is anticipated that additional agents will become available to potentiate TKI therapy and/or circumvent TKI resistance in Ph+ ALL. Recent genomic studies have identified a subtype of high-risk pediatric B-cell-precursor ALL with a gene-expression profile similar to that of Ph+ ALL, suggestive of active kinase signaling. Many of these Ph-like ALL cases harbor chromosome rearrangements and mutations that dysregulate cytokine receptor and kinase signaling, and these leukemias may also be candidates for TKI therapy.

Resistance Profiling of BCR-ABL Compound Mutations Linked to Tyrosine Kinase Inhibitor Therapy Failure in Chronic Myeloid Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1416-1416 ◽  
Author(s):  
Christopher A. Eide ◽  
Matthew S. Zabriskie ◽  
Lauren T. Adrian ◽  
Thoralf Lange ◽  
Michael W. Deininger ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Point Mutations ◽  
Patient Costs ◽  
Clinical Resistance ◽  
Data Manager

Abstract Abstract 1416 Among patients with chronic myeloid leukemia (CML) who develop resistance to ABL tyrosine kinase inhibitor (TKI) therapy, the most common explanation is acquisition of point mutations within the BCR-ABL kinase domain that interfere with drug binding. The second-generation ABL TKIs nilotinib and dasatinib have proven effective against most imatinib-resistant mutants, with the critical exception of BCR-ABLT315I. Furthermore, sequential treatment with ABL TKIs can select for BCR-ABL compound mutations (two or more point mutations within the same BCR-ABL molecule) that are resistant to multiple inhibitors. With third-generation ABLT315I TKIs such as ponatinib (AP24534) and DCC-2036 currently in clinical development, containment of single point mutation-based resistance appears tenable. However, the ability of available inhibitors to address resistance due to the growing number of reported BCR-ABL compound mutations has not been investigated. Here, we generated stable Ba/F3 cell lines expressing a panel of clinically-observed BCR-ABL compound mutants (including two novel mutants which we identified from CML clinical resistance specimens, BCR-ABLG250E/V299L and BCR-ABLT315I/H396R) as well as cells expressing each constituent mutation. Cells were plated in graded concentrations of imatinib, nilotinib, dasatinib, ponatinib, and DCC-2036, incubated for 72 hours, and cellular proliferation was analyzed by standard methanethiosulfonate-based assay. We found that, consistent with our previous cell-based mutagenesis screens for resistance (Blood 2006, 108: 2332–8; Cancer Cell 2009, 16: 401–12; Cancer Research 2011, 71: 3189–95), all five tested inhibitors demonstrated unique but partially overlapping resistance profiles relative to the panel of compound mutants tested. As expected, all mutants harboring a T315I component were insensitive to imatinib, nilotinib, and dasatinib, whereas ponatinib and DCC-2036 showed varying levels of efficacy against such mutants. The most resistant, clinically reported compound mutants were BCR-ABLG250E/T315I, BCR-ABLE255K/T315I, and BCR-ABLE255V/T315I, all of which conferred markedly increased resistance to ponatinib and DCC-2036 relative to cells expressing each constituent mutation alone (Table 1). For example, the most resistant compound mutant, BCR-ABLE255V/T315I, conferred high-level resistance to ponatinib and DCC-2036 (IC50: 425 and 1272 nM, respectively), while cells expressing either BCR-ABLE255V or BCR-ABLT315I were inhibited at clinically relevant concentrations of ponatinib (IC50: 33 and 18 nM, respectively) or DCC-2036 (IC50: 659 and 149 nM, respectively). Additional structural, biochemical, and signaling pathway analyses addressing the striking differences in sensitivity of BCR-ABL compound mutants versus their constituent mutations are being pursued and will be presented. Overall, our findings demonstrate that BCR-ABL compound mutations confer varying degrees of resistance to currently available ABL TKIs, necessitating rational treatment selection to optimize outcomes. Patients harboring highly multi-drug-resistant compound mutants such as BCR-ABLE255V/T315I may have limited therapeutic options available, warranting investigation into combination therapies involving ABLT315I TKIs. Furthermore, studies of compound mutation-based resistance mechanisms in CML advance the possibility of maximum disease control in a greater proportion of patients and have important implications for anticipating resistance and designing treatment strategies in similar, more rapid clinical resistance scenarios such as those encountered with EGFR or ALK mutations in non-small-cell lung cancer.Table 1.Cell proliferation IC 50s for clinically-observed BCR-ABL compound mutants.Cell lineimatinib (nM)nilotinib (nM)dasatinib (nM)Ponatinib (nM)DCC-2036 (nM)Parental>5120>5120>768>768>5120Native BCR-ABL270121.22.337BCR-ABLG250E26391702.87.7180BCR-ABLG250E/T315I>5120>5120>76849627BCR-ABLE255K>51203268.923410BCR-ABLE255K/T315I>5120>5120>768106585BCR-ABLE255V>512011771333659BCR-ABLE255V/T315I>5120>5120>7684251272BCR-ABLT315I>5120>5120>76818149 Disclosures: Druker: Novartis: OHSU has clinical trial contracts with Novartis to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; Bristol-Myers-Squibb: OHSU has clinical trial contracts with Bristol-Myers-Squibb to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; MolecularMD: OHSU and Dr. Druker have a financial interest in MolecularMD. Technology used in this research has been licensed to MolecularMD. This potential COI has been reviewed and managed by the OHSU COI in Research Committee & Integrity Program Oversight Council.

scholarly journals Proteasome Regulation by Reversible Tyrosine Phosphorylation at the Membrane

2020 ◽  
Author(s):  
Lu Chen ◽  
Xin Shu ◽  
Qiong Chen ◽  
Tiantian Wei ◽  
Xiaorong Wang ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Tyrosine Phosphatase ◽  
Tumor Model ◽  
26S Proteasome ◽  
Cancer Therapies ◽  
Proteome Level ◽  
Proteasome Regulation

AbstractReversible phosphorylation has emerged as an important mechanism for regulating 26S proteasome function in health and disease. Over 100 phospho-tyrosine (pTyr) sites of the human proteasome have been detected, and yet their function and regulation remain poorly understood. Here we show that the 19S subunit Rpt2 is phosphorylated at Tyr439, a strictly conserved residue within the C-terminal HbYX motif of Rpt2 that is essential for 26S proteasome assembly. Unexpectedly, we found that Y439 phosphorylation depends on Rpt2 membrane localization mediated by its N-myristoylation. Multiple receptor tyrosine kinases (RTKs) can trigger Rpt2-Y439 phosphorylation by activating Src, a N-myristoylated tyrosine kinase. Src directly phosphorylates Rpt2-Y439 in vitro and negatively regulates 26S proteasome integrity and activity at cellular membranes, which can be reversed by the membrane-associated isoform of protein tyrosine phosphatase non-receptor type 2 (PTPN2). In H1975 lung cancer cells with activated Src, blocking Rpt2-Y439 phosphorylation by the Y439F mutation conferred partial resistance to the Src inhibitor saracatinib both in vitro and in a mouse xenograft tumor model, and caused significant changes of cellular responses to saracatinib at the proteome level. Our study has defined a novel mechanism involved in the spatial regulation of proteasome function and provided new insights into tyrosine kinase inhibitor-based anti-cancer therapies.

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