Targeting key survival signaling pathways for the treatment of leukemia

Research output: ThesisDoctoral Thesis

Abstract

Leukemia is a cancer of the blood cells that has traditionally been treated with unselective chemotherapeutic agents. Advances in molecular biology and sequencing technologies have revolutionized the understanding of molecular and genetic factors in leukemia pathogenesis. Each leukemia patient harbors a unique set of genetic abnormalities, which result in impaired regulation of cell proliferation and differentiation. Increased cell proliferation is commonly mediated through overactive signaling cascades, such as JAK/STAT, PI3K/AKT/mTOR and MAPK pathways. The improved understanding of the molecular pathobiology has led to the development of small molecule inhibitors that can directly bind to target proteins and inhibit aberrant signaling. However, the molecular landscape of rare types of leukemia has not been comprehensively studied and our understanding of the relationship between cancer genotype, phenotype and drug function is limited. In study I, we aimed to identify novel driver mutations in large granular lymphocyte (LGL) leukemia. Earlier, our research group discovered that 40% of LGL leukemia patients had activating point mutations in the STAT3 gene. By using exome and targeted sequencing we identified 4/211 patients to carry an activating STAT5B mutation in the SH2 domain (N642H and Y640F). In study II, we identified STAT5B mutations in 6/68 (8%) T-cell acute lymphoblastic leukemia (T-ALL) patients. The finding represents a novel mechanism leading to the activation of the IL7R/JAK/STAT5 pathway. Our initial results also suggest that hyperactive STAT5B might be involved in the overexpression of anti-apoptotic BCL-xL. In study III, using a functional reporter assay we screened 306 oncology compounds to find potential hits that can decrease the cellular activity of mutant STAT3. The most potent targeted compounds inhibiting STAT3 activity were cyclin-dependent kinase (CDK), mammalian target of rapamycin (mTOR), heat shock protein 90 (Hsp90), and Janus kinase (JAK) inhibitors. Among these compounds only the Hsp90 inhibitors effectively inhibited both the mutant and wild type STAT3 phosphorylation and activity. In study IV, we developed a flow cytometry-based drug screening assay to assess cell population specific drug responses in heterogenous AML samples. Using the assay, we were able to simultaneously measure the drug responses of leukemic blasts, more mature leukemic cells and lymphocytes. The data showed that targeted therapies have different efficacies towards leukemic AML cells at distinct stages of myeloid differentiation. Particularly, BCL2 inhibitor venetoclax was toxic to immature blasts but was not effective against more differentiated monocytic and granulopoietic cells. In conclusion, we identified novel STAT5B mutations in two rare lymphoproliferative diseases, T-ALL and LGL leukemia. Furthermore, we demonstrate that our ex vivo flow cytometry-based drug screening platform can increase the functional understanding of drug effects.
Original languageEnglish
Place of PublicationHelsinki
Publisher
Print ISBNs978-951-51-4448-5
Electronic ISBNs978-951-51-4449-2
Publication statusPublished - 2018
MoE publication typeG5 Doctoral dissertation (article)

Fields of Science

  • Leukemia, T-Cell
  • +genetics
  • Leukemia, Large Granular Lymphocytic
  • Precursor T-Cell Lymphoblastic Leukemia-Lymphoma
  • STAT3 Transcription Factor
  • STAT5 Transcription Factor
  • Flow Cytometry
  • Sulfonamides
  • Bridged Bicyclo Compounds, Heterocyclic
  • Antineoplastic Agents
  • Drug Evaluation, Preclinical
  • Cell Transformation, Neoplastic
  • Cyclin-Dependent Kinases
  • TOR Serine-Threonine Kinases
  • HSP90 Heat-Shock Proteins
  • Janus Kinase Inhibitors
  • Phosphatidylinositol 3-Kinases
  • Proto-Oncogene Proteins c-akt
  • Signal Transduction
  • Sirolimus
  • src Homology Domains
  • 3122 Cancers
  • 1184 Genetics, developmental biology, physiology
  • 3111 Biomedicine

Cite this

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title = "Targeting key survival signaling pathways for the treatment of leukemia",
abstract = "Leukemia is a cancer of the blood cells that has traditionally been treated with unselective chemotherapeutic agents. Advances in molecular biology and sequencing technologies have revolutionized the understanding of molecular and genetic factors in leukemia pathogenesis. Each leukemia patient harbors a unique set of genetic abnormalities, which result in impaired regulation of cell proliferation and differentiation. Increased cell proliferation is commonly mediated through overactive signaling cascades, such as JAK/STAT, PI3K/AKT/mTOR and MAPK pathways. The improved understanding of the molecular pathobiology has led to the development of small molecule inhibitors that can directly bind to target proteins and inhibit aberrant signaling. However, the molecular landscape of rare types of leukemia has not been comprehensively studied and our understanding of the relationship between cancer genotype, phenotype and drug function is limited. In study I, we aimed to identify novel driver mutations in large granular lymphocyte (LGL) leukemia. Earlier, our research group discovered that 40{\%} of LGL leukemia patients had activating point mutations in the STAT3 gene. By using exome and targeted sequencing we identified 4/211 patients to carry an activating STAT5B mutation in the SH2 domain (N642H and Y640F). In study II, we identified STAT5B mutations in 6/68 (8{\%}) T-cell acute lymphoblastic leukemia (T-ALL) patients. The finding represents a novel mechanism leading to the activation of the IL7R/JAK/STAT5 pathway. Our initial results also suggest that hyperactive STAT5B might be involved in the overexpression of anti-apoptotic BCL-xL. In study III, using a functional reporter assay we screened 306 oncology compounds to find potential hits that can decrease the cellular activity of mutant STAT3. The most potent targeted compounds inhibiting STAT3 activity were cyclin-dependent kinase (CDK), mammalian target of rapamycin (mTOR), heat shock protein 90 (Hsp90), and Janus kinase (JAK) inhibitors. Among these compounds only the Hsp90 inhibitors effectively inhibited both the mutant and wild type STAT3 phosphorylation and activity. In study IV, we developed a flow cytometry-based drug screening assay to assess cell population specific drug responses in heterogenous AML samples. Using the assay, we were able to simultaneously measure the drug responses of leukemic blasts, more mature leukemic cells and lymphocytes. The data showed that targeted therapies have different efficacies towards leukemic AML cells at distinct stages of myeloid differentiation. Particularly, BCL2 inhibitor venetoclax was toxic to immature blasts but was not effective against more differentiated monocytic and granulopoietic cells. In conclusion, we identified novel STAT5B mutations in two rare lymphoproliferative diseases, T-ALL and LGL leukemia. Furthermore, we demonstrate that our ex vivo flow cytometry-based drug screening platform can increase the functional understanding of drug effects.",
keywords = "Leukemia, T-Cell, +genetics, Leukemia, Large Granular Lymphocytic, Precursor T-Cell Lymphoblastic Leukemia-Lymphoma, STAT3 Transcription Factor, STAT5 Transcription Factor, Flow Cytometry, Sulfonamides, Bridged Bicyclo Compounds, Heterocyclic, Antineoplastic Agents, Drug Evaluation, Preclinical, Cell Transformation, Neoplastic, Cyclin-Dependent Kinases, TOR Serine-Threonine Kinases, HSP90 Heat-Shock Proteins, Janus Kinase Inhibitors, Phosphatidylinositol 3-Kinases, Proto-Oncogene Proteins c-akt, Signal Transduction, Sirolimus, src Homology Domains, 3122 Cancers, 1184 Genetics, developmental biology, physiology, 3111 Biomedicine",
author = "Heikki Kuusanm{\"a}ki",
note = "M1 - 88 s. + liitteet",
year = "2018",
language = "English",
isbn = "978-951-51-4448-5",
publisher = "[H. Kuusanm{\"a}ki]",
address = "Finland",

}

Targeting key survival signaling pathways for the treatment of leukemia. / Kuusanmäki, Heikki.

Helsinki : [H. Kuusanmäki], 2018. 88 p.

Research output: ThesisDoctoral Thesis

TY - THES

T1 - Targeting key survival signaling pathways for the treatment of leukemia

AU - Kuusanmäki, Heikki

N1 - M1 - 88 s. + liitteet

PY - 2018

Y1 - 2018

N2 - Leukemia is a cancer of the blood cells that has traditionally been treated with unselective chemotherapeutic agents. Advances in molecular biology and sequencing technologies have revolutionized the understanding of molecular and genetic factors in leukemia pathogenesis. Each leukemia patient harbors a unique set of genetic abnormalities, which result in impaired regulation of cell proliferation and differentiation. Increased cell proliferation is commonly mediated through overactive signaling cascades, such as JAK/STAT, PI3K/AKT/mTOR and MAPK pathways. The improved understanding of the molecular pathobiology has led to the development of small molecule inhibitors that can directly bind to target proteins and inhibit aberrant signaling. However, the molecular landscape of rare types of leukemia has not been comprehensively studied and our understanding of the relationship between cancer genotype, phenotype and drug function is limited. In study I, we aimed to identify novel driver mutations in large granular lymphocyte (LGL) leukemia. Earlier, our research group discovered that 40% of LGL leukemia patients had activating point mutations in the STAT3 gene. By using exome and targeted sequencing we identified 4/211 patients to carry an activating STAT5B mutation in the SH2 domain (N642H and Y640F). In study II, we identified STAT5B mutations in 6/68 (8%) T-cell acute lymphoblastic leukemia (T-ALL) patients. The finding represents a novel mechanism leading to the activation of the IL7R/JAK/STAT5 pathway. Our initial results also suggest that hyperactive STAT5B might be involved in the overexpression of anti-apoptotic BCL-xL. In study III, using a functional reporter assay we screened 306 oncology compounds to find potential hits that can decrease the cellular activity of mutant STAT3. The most potent targeted compounds inhibiting STAT3 activity were cyclin-dependent kinase (CDK), mammalian target of rapamycin (mTOR), heat shock protein 90 (Hsp90), and Janus kinase (JAK) inhibitors. Among these compounds only the Hsp90 inhibitors effectively inhibited both the mutant and wild type STAT3 phosphorylation and activity. In study IV, we developed a flow cytometry-based drug screening assay to assess cell population specific drug responses in heterogenous AML samples. Using the assay, we were able to simultaneously measure the drug responses of leukemic blasts, more mature leukemic cells and lymphocytes. The data showed that targeted therapies have different efficacies towards leukemic AML cells at distinct stages of myeloid differentiation. Particularly, BCL2 inhibitor venetoclax was toxic to immature blasts but was not effective against more differentiated monocytic and granulopoietic cells. In conclusion, we identified novel STAT5B mutations in two rare lymphoproliferative diseases, T-ALL and LGL leukemia. Furthermore, we demonstrate that our ex vivo flow cytometry-based drug screening platform can increase the functional understanding of drug effects.

AB - Leukemia is a cancer of the blood cells that has traditionally been treated with unselective chemotherapeutic agents. Advances in molecular biology and sequencing technologies have revolutionized the understanding of molecular and genetic factors in leukemia pathogenesis. Each leukemia patient harbors a unique set of genetic abnormalities, which result in impaired regulation of cell proliferation and differentiation. Increased cell proliferation is commonly mediated through overactive signaling cascades, such as JAK/STAT, PI3K/AKT/mTOR and MAPK pathways. The improved understanding of the molecular pathobiology has led to the development of small molecule inhibitors that can directly bind to target proteins and inhibit aberrant signaling. However, the molecular landscape of rare types of leukemia has not been comprehensively studied and our understanding of the relationship between cancer genotype, phenotype and drug function is limited. In study I, we aimed to identify novel driver mutations in large granular lymphocyte (LGL) leukemia. Earlier, our research group discovered that 40% of LGL leukemia patients had activating point mutations in the STAT3 gene. By using exome and targeted sequencing we identified 4/211 patients to carry an activating STAT5B mutation in the SH2 domain (N642H and Y640F). In study II, we identified STAT5B mutations in 6/68 (8%) T-cell acute lymphoblastic leukemia (T-ALL) patients. The finding represents a novel mechanism leading to the activation of the IL7R/JAK/STAT5 pathway. Our initial results also suggest that hyperactive STAT5B might be involved in the overexpression of anti-apoptotic BCL-xL. In study III, using a functional reporter assay we screened 306 oncology compounds to find potential hits that can decrease the cellular activity of mutant STAT3. The most potent targeted compounds inhibiting STAT3 activity were cyclin-dependent kinase (CDK), mammalian target of rapamycin (mTOR), heat shock protein 90 (Hsp90), and Janus kinase (JAK) inhibitors. Among these compounds only the Hsp90 inhibitors effectively inhibited both the mutant and wild type STAT3 phosphorylation and activity. In study IV, we developed a flow cytometry-based drug screening assay to assess cell population specific drug responses in heterogenous AML samples. Using the assay, we were able to simultaneously measure the drug responses of leukemic blasts, more mature leukemic cells and lymphocytes. The data showed that targeted therapies have different efficacies towards leukemic AML cells at distinct stages of myeloid differentiation. Particularly, BCL2 inhibitor venetoclax was toxic to immature blasts but was not effective against more differentiated monocytic and granulopoietic cells. In conclusion, we identified novel STAT5B mutations in two rare lymphoproliferative diseases, T-ALL and LGL leukemia. Furthermore, we demonstrate that our ex vivo flow cytometry-based drug screening platform can increase the functional understanding of drug effects.

KW - Leukemia, T-Cell

KW - +genetics

KW - Leukemia, Large Granular Lymphocytic

KW - Precursor T-Cell Lymphoblastic Leukemia-Lymphoma

KW - STAT3 Transcription Factor

KW - STAT5 Transcription Factor

KW - Flow Cytometry

KW - Sulfonamides

KW - Bridged Bicyclo Compounds, Heterocyclic

KW - Antineoplastic Agents

KW - Drug Evaluation, Preclinical

KW - Cell Transformation, Neoplastic

KW - Cyclin-Dependent Kinases

KW - TOR Serine-Threonine Kinases

KW - HSP90 Heat-Shock Proteins

KW - Janus Kinase Inhibitors

KW - Phosphatidylinositol 3-Kinases

KW - Proto-Oncogene Proteins c-akt

KW - Signal Transduction

KW - Sirolimus

KW - src Homology Domains

KW - 3122 Cancers

KW - 1184 Genetics, developmental biology, physiology

KW - 3111 Biomedicine

M3 - Doctoral Thesis

SN - 978-951-51-4448-5

PB - [H. Kuusanmäki]

CY - Helsinki

ER -