A Modified Form of Diphthamide Causes Immunotoxin Resistance in a Lymphoma Cell Line with a Deletion of the WDR85 Gene

Wei, Hui; Bera, Tapan K.; Wayne, Alan S.; Xiang, Laiman; Colantonio, Simona; Chertov, Oleg; Pastan, Ira

doi:10.1074/jbc.m113.461343

Cited by 29 publications

(34 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This insensitivity may reflect innate resistance based on the lack of CMML cell proliferation ex vivo, as described previously for constructs based on diphtheria toxin 48 or for constructs based on Pseudomonas exotoxin A. 49 We also investigated the relationship between the cytotoxicity of the PI9-sensitive and PI9-resistant constructs and the abundance of PI9 immediately after the isolation of cells from peripheral blood and again after cultivation for different time intervals. Western blots of all samples showed minimal signals just after isolation but strong bands during cultivation, suggesting that PI9 is upregulated in culture.…”

Section: Discussionmentioning

confidence: 99%

Targeted ex vivo reduction of CD64‐positive monocytes in chronic myelomonocytic leukemia and acute myelomonocytic leukemia using human granzyme B‐based cytolytic fusion proteins

Schiffer

Rosinke

Jost

et al. 2014

Intl Journal of Cancer

View full text Add to dashboard Cite

CMML (chronic myelomonocytic leukemia) belongs to the group of myeloid neoplasms known as myelodysplastic and myeloproliferative diseases. In some patients with a history of CMML, the disease transforms to acute myelomonocytic leukemia (AMML). There are no specific treatment options for patients suffering from CMML except for supportive care and DNA methyltransferase inhibitors in patients with advanced disease. New treatment strategies are urgently required, so we have investigated the use of immunotherapeutic directed cytolytic fusion proteins (CFPs), which are chimeric proteins comprising a selective domain and a toxic component (preferably of human origin to avoid immunogenicity). The human serine protease granzyme B is a prominent candidate for tumor immunotherapy because it is expressed in cytotoxic T lymphocytes and natural killer cells. Here, we report the use of CD64 as a novel target for specific CMML and AMML therapy, and correlate CD64 expression with typical surface markers representing these diseases. We demonstrate that CD64-specific human CFPs kill CMML and AMML cells ex vivo, and that the mutant granzyme B protein R201K is more cytotoxic than the wild-type enzyme in the presence of the granzyme B inhibitor PI9. Besides, the human CFP based on the granzyme B mutant was also able to kill AMML or CMML probes resistant to Pseudomonas exotoxin A.The clonal hematopoietic stem cell disorder CMML (chronic myelomonocytic leukemia), is a highly aggressive and resistant form of leukemia. It is characterized by persistent monocytosis in the peripheral blood, at least one dysplasia component in the bone marrow, and <20% promonocytes and blasts in peripheral blood and bone marrow. 1 CMML can be classified further according to blast numbers, that is, CMML1 (<5% blasts in peripheral blood and <10% blasts in bone marrow) and CMML2 (<20% blasts in peripheral blood and 10-19% blasts in bone marrow).2,3 The disease occurs mainly in elderly people with a median survival of 15-20 months and leukemic transformation rates of 15-30%, both factors depending on the disease subtype. 4 Other diagnostic criteria include immunophenotyping based on the overlapping antigen expression patterns in CMML and acute myelomonocytic leukemia (AMML). The expression of CD56 combined with reductions in the levels of myeloid markers such as CD15 and CD13 is a unique signature of CMML monocytes.5 Furthermore, CD14 is expressed at higher levels on bone marrow monocytes in CMML compared with reactive monocytosis and normal marrow samples. 5 High levels of CD33 have also been reported. 6 Clonal cytogenetic abnormalities are found in 20-40% of CMML patients, but these do not appear to be specific. [7][8][9] The close relationship between CMML and other myelodysplastic (MDS)/MPNs makes correct diagnosis and treatment a significant challenge. Novel molecular markers described more recently include specific alleles of TET2 and CBL, but these

show abstract

Section: Discussionmentioning

confidence: 99%

Targeted ex vivo reduction of CD64‐positive monocytes in chronic myelomonocytic leukemia and acute myelomonocytic leukemia using human granzyme B‐based cytolytic fusion proteins

Schiffer

Rosinke

Jost

et al. 2014

Intl Journal of Cancer

View full text Add to dashboard Cite

show abstract

“…However, the "complete picture" is (with the exception of the yeast pathway) to a large portion is composed of observations made in different cell types on single genes. Many reports related to diphthamide synthesis of mammalian cells describe "partial knockouts" and "partial phenotypes" (i.e., reduced levels but not complete loss of diphthamide modification or toxin sensitivities) (7)(8)(9) So far, the function of diphthamide on eEF2 also remained rather elusive. Reports indicate that it contributes to translation fidelity (10)(11)(12)(13).…”

mentioning

confidence: 99%

Loss of diphthamide pre-activates NF-κB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor

Stahl¹,

Seidl²,

Ducret

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

The diphthamide on human eukaryotic translation elongation factor 2 (eEF2) is the target of ADP ribosylating diphtheria toxin (DT) and Pseudomonas exotoxin A (PE). This modification is synthesized by seven dipthamide biosynthesis proteins (DPH1-DPH7) and is conserved among eukaryotes and archaea. We generated MCF7 breast cancer cell line-derived DPH gene knockout (ko) cells to assess the impact of complete or partial inactivation on diphthamide synthesis and toxin sensitivity, and to address the biological consequence of diphthamide deficiency. Cells with heterozygous gene inactivation still contained predominantly diphthamide-modified eEF2 and were as sensitive to PE and DT as parent cells. Thus, DPH gene copy number reduction does not affect overall diphthamide synthesis and toxin sensitivity. Complete inactivation of DPH1, DPH2, DPH4, and DPH5 generated viable cells without diphthamide. DPH1ko, DPH2ko, and DPH4ko harbored unmodified eEF2 and DPH5ko ACP-(diphthine-precursor) modified eEF2. Loss of diphthamide prevented ADP ribosylation of eEF2, rendered cells resistant to PE and DT, but does not affect sensitivity toward other protein synthesis inhibitors, such as saporin or cycloheximide. Surprisingly, cells without diphthamide (independent of which the DPH gene compromised) were presensitized toward nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) and death-receptor pathways without crossing lethal thresholds. In consequence, loss of diphthamide rendered cells hypersensitive toward TNF-mediated apoptosis. This finding suggests a role of diphthamide in modulating NF-κB, death receptor, or apoptosis pathways.ADP-ribosylation of eEF2 | Pseudomonas exotoxin | diphtheria toxin | translation | DPH gene knockout

show abstract

“…***P ≤ 0Á001 compared to the buffer control. candidates, which exploit different kinds of apoptotic pathways (Wei et al, 2013). This would finally enable choice when selecting the most promising combination of a diseases-specific ligand and an effector lead agent.…”

Section: Discussionmentioning

confidence: 99%

Human microtubule‐associated protein tau mediates targeted killing of CD30⁺ lymphoma cells in vitro and inhibits tumour growth in vivo

et al. 2013

View full text Add to dashboard Cite

SummaryHodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (sALCL) are rare lymphoproliferative cancer types. Although most HL patients can be cured by chemo-and radio-therapy, 4-50% of patients relapse and have a poor prognosis. The need for improved therapeutic options for patients with relapsed or refractory disease has been addressed by CD30-specific antibody-based immunotherapeutics. However, available CD30-specific monoclonal antibodies (mAbs), antibody drug conjugates (ADCs) or chimeric immunotoxins suffer from the requirement of a functional host immunity, undesirable immune reactions or heterogeneity and instability, respectively. Here, we present a new fusion protein comprised of the CD30-specific antibody single-chain fragment Ki4(scFv) and the human pro-apoptotic effector protein, microtubule-associated protein tau (MAPT). Ki4(scFv)-MAP selectively induced apoptosis in rapidly proliferating L540cy, L428, and Karpas 299 cells in a dose-dependent manner. Tubulin polymerization assays confirmed that Ki4(scFv)-MAP stabilizes microtubules, suggesting a mechanism for its pro-apoptotic action. Dosefinding experiments proved that Ki4(scFv)-MAP is well tolerated in mice compared to the previously reported Ki4(scFv)-ETA'. Ki4(scFv)-MAP significantly inhibited growth of subcutaneous L540cy xenograft tumours in mice. Our data present a novel approach for the treatment of CD30 + lymphomas, combining the binding specificity of a target-specific antibody fragment with the selective cytotoxicity of MAPT towards proliferating lymphoma cells.

show abstract

A Modified Form of Diphthamide Causes Immunotoxin Resistance in a Lymphoma Cell Line with a Deletion of the WDR85 Gene

Cited by 29 publications

References 30 publications

Targeted ex vivo reduction of CD64‐positive monocytes in chronic myelomonocytic leukemia and acute myelomonocytic leukemia using human granzyme B‐based cytolytic fusion proteins

Targeted ex vivo reduction of CD64‐positive monocytes in chronic myelomonocytic leukemia and acute myelomonocytic leukemia using human granzyme B‐based cytolytic fusion proteins

Loss of diphthamide pre-activates NF-κB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor

Human microtubule‐associated protein tau mediates targeted killing of CD30⁺ lymphoma cells in vitro and inhibits tumour growth in vivo

Contact Info

Product

Resources

About

A Modified Form of Diphthamide Causes Immunotoxin Resistance in a Lymphoma Cell Line with a Deletion of the WDR85 Gene

Cited by 29 publications

References 30 publications

Targeted ex vivo reduction of CD64‐positive monocytes in chronic myelomonocytic leukemia and acute myelomonocytic leukemia using human granzyme B‐based cytolytic fusion proteins

Targeted ex vivo reduction of CD64‐positive monocytes in chronic myelomonocytic leukemia and acute myelomonocytic leukemia using human granzyme B‐based cytolytic fusion proteins

Loss of diphthamide pre-activates NF-κB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor

Human microtubule‐associated protein tau mediates targeted killing of CD30+ lymphoma cells in vitro and inhibits tumour growth in vivo

Contact Info

Product

Resources

About

Human microtubule‐associated protein tau mediates targeted killing of CD30⁺ lymphoma cells in vitro and inhibits tumour growth in vivo