Synergistic Cytotoxic Effect of Busulfan and the PARP Inhibitor Veliparib in Myeloproliferative Neoplasms

Pritesh R. Patel, Vitalyi Senyuk, Natalie S. Rodriguez, Annie L. Oh, Elisa Bonetti, Dolores Mahmud, Gianni Barosi, Nadim Mahmud, Damiano Rondelli

Research output: Contribution to journalArticle

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Abstract

Patients with high-risk myeloproliferative neoplasms (MPNs), and in particular myelofibrosis (MF), can be cured only with allogeneic hematopoietic stem cell transplantation (HSCT). Because MPNs and JAK2 V617F -mutated cells show genomic instability, stalled replication forks, and baseline DNA double-strand breaks, DNA repair inhibition with poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors represents a potential novel therapy. Because the alkylating agent busulfan is integral in conditioning regimens for HSCT and leads to stalled replication forks through DNA strand cross-linking, we hypothesized that PARP inhibition with veliparib in combination with busulfan may lead to synergistic cytotoxicity in MPN cells. We first treated 2 MPN cell lines harboring the JAK2 V617F mutation (SET2 and HEL) with veliparib at increasing concentrations and measured cell proliferation. SET2 and HEL cells were relatively sensitive to veliparib (IC 50 of 11.3 μM and 74.2 μM, respectively). We next treated cells with increasing doses of busulfan in combination with 4 μM veliparib and found that the busulfan IC 50 decreased from 27 μM to 4 μM in SET2 cells and from 45.1 μM to 28.1 μM in HEL cells. The mean combination index was.55 for SET2 cells and.40 for HEL cells. Combination treatment of SET2 cells caused G2M arrest in 53% of cells, compared with 30% with veliparib alone and 35% with busulfan alone. G2M arrest was associated with activation of the ATR-Chk1 pathway, as shown by an immunofluorescence assay for phosphorylated Chk1 (p-Chk1). We then tested in vivo the effect of combined low doses of busulfan and veliparib in a JAK2 V617F MPN-AML xenotransplant model. Vehicle- and veliparib-treated mice had similar median survival of 39 and 40 days, respectively. Combination treatment increased median survival from 47 days (busulfan alone) to 50 days (P =.02). Finally, we tested the combined effect of busulfan and veliparib on CD34 + cells obtained from the bone marrow or peripheral blood of 5 patients with JAK2 V617F -mutated and 2 patients with CALR-mutated MF. MF cells treated with the combination of veliparib and busulfan showed reduced colony formation compared with busulfan alone (87% versus 68%; P =.001). In contrast, treatment of normal CD34 + cells with veliparib did not affect colony growth. Here we show that in vivo confirmation that treatment with the PARP-1 inhibitor veliparib and busulfan results in synergistic cytotoxicity in MPN cells. Our data provide the rationale for testing novel pretransplantation conditioning regimens with combinations of PARP-1 inhibition and reduced doses of alkylators, such as busulfan and melphalan, for high-risk MPNs or MPN-derived acute myelogenous leukemia.

Original languageEnglish
JournalBiology of Blood and Marrow Transplantation
DOIs
Publication statusPublished - Jan 1 2019

Fingerprint

Busulfan
Neoplasms
Primary Myelofibrosis
Alkylating Agents
Hematopoietic Stem Cell Transplantation
veliparib
Poly(ADP-ribose) Polymerase Inhibitors
Therapeutics
Melphalan
Survival
Double-Stranded DNA Breaks
Genomic Instability
Acute Myeloid Leukemia
DNA Repair
Bone Marrow Cells
Fluorescent Antibody Technique

Keywords

  • Alkylator chemotherapy
  • Busulfan
  • Myeloproliferative neoplasms
  • PARP-1
  • Veliparib

ASJC Scopus subject areas

  • Hematology
  • Transplantation

Cite this

Synergistic Cytotoxic Effect of Busulfan and the PARP Inhibitor Veliparib in Myeloproliferative Neoplasms. / Patel, Pritesh R.; Senyuk, Vitalyi; Rodriguez, Natalie S.; Oh, Annie L.; Bonetti, Elisa; Mahmud, Dolores; Barosi, Gianni; Mahmud, Nadim; Rondelli, Damiano.

In: Biology of Blood and Marrow Transplantation, 01.01.2019.

Research output: Contribution to journalArticle

Patel, Pritesh R. ; Senyuk, Vitalyi ; Rodriguez, Natalie S. ; Oh, Annie L. ; Bonetti, Elisa ; Mahmud, Dolores ; Barosi, Gianni ; Mahmud, Nadim ; Rondelli, Damiano. / Synergistic Cytotoxic Effect of Busulfan and the PARP Inhibitor Veliparib in Myeloproliferative Neoplasms. In: Biology of Blood and Marrow Transplantation. 2019.
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abstract = "Patients with high-risk myeloproliferative neoplasms (MPNs), and in particular myelofibrosis (MF), can be cured only with allogeneic hematopoietic stem cell transplantation (HSCT). Because MPNs and JAK2 V617F -mutated cells show genomic instability, stalled replication forks, and baseline DNA double-strand breaks, DNA repair inhibition with poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors represents a potential novel therapy. Because the alkylating agent busulfan is integral in conditioning regimens for HSCT and leads to stalled replication forks through DNA strand cross-linking, we hypothesized that PARP inhibition with veliparib in combination with busulfan may lead to synergistic cytotoxicity in MPN cells. We first treated 2 MPN cell lines harboring the JAK2 V617F mutation (SET2 and HEL) with veliparib at increasing concentrations and measured cell proliferation. SET2 and HEL cells were relatively sensitive to veliparib (IC 50 of 11.3 μM and 74.2 μM, respectively). We next treated cells with increasing doses of busulfan in combination with 4 μM veliparib and found that the busulfan IC 50 decreased from 27 μM to 4 μM in SET2 cells and from 45.1 μM to 28.1 μM in HEL cells. The mean combination index was.55 for SET2 cells and.40 for HEL cells. Combination treatment of SET2 cells caused G2M arrest in 53{\%} of cells, compared with 30{\%} with veliparib alone and 35{\%} with busulfan alone. G2M arrest was associated with activation of the ATR-Chk1 pathway, as shown by an immunofluorescence assay for phosphorylated Chk1 (p-Chk1). We then tested in vivo the effect of combined low doses of busulfan and veliparib in a JAK2 V617F MPN-AML xenotransplant model. Vehicle- and veliparib-treated mice had similar median survival of 39 and 40 days, respectively. Combination treatment increased median survival from 47 days (busulfan alone) to 50 days (P =.02). Finally, we tested the combined effect of busulfan and veliparib on CD34 + cells obtained from the bone marrow or peripheral blood of 5 patients with JAK2 V617F -mutated and 2 patients with CALR-mutated MF. MF cells treated with the combination of veliparib and busulfan showed reduced colony formation compared with busulfan alone (87{\%} versus 68{\%}; P =.001). In contrast, treatment of normal CD34 + cells with veliparib did not affect colony growth. Here we show that in vivo confirmation that treatment with the PARP-1 inhibitor veliparib and busulfan results in synergistic cytotoxicity in MPN cells. Our data provide the rationale for testing novel pretransplantation conditioning regimens with combinations of PARP-1 inhibition and reduced doses of alkylators, such as busulfan and melphalan, for high-risk MPNs or MPN-derived acute myelogenous leukemia.",
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T1 - Synergistic Cytotoxic Effect of Busulfan and the PARP Inhibitor Veliparib in Myeloproliferative Neoplasms

AU - Patel, Pritesh R.

AU - Senyuk, Vitalyi

AU - Rodriguez, Natalie S.

AU - Oh, Annie L.

AU - Bonetti, Elisa

AU - Mahmud, Dolores

AU - Barosi, Gianni

AU - Mahmud, Nadim

AU - Rondelli, Damiano

PY - 2019/1/1

Y1 - 2019/1/1

N2 - Patients with high-risk myeloproliferative neoplasms (MPNs), and in particular myelofibrosis (MF), can be cured only with allogeneic hematopoietic stem cell transplantation (HSCT). Because MPNs and JAK2 V617F -mutated cells show genomic instability, stalled replication forks, and baseline DNA double-strand breaks, DNA repair inhibition with poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors represents a potential novel therapy. Because the alkylating agent busulfan is integral in conditioning regimens for HSCT and leads to stalled replication forks through DNA strand cross-linking, we hypothesized that PARP inhibition with veliparib in combination with busulfan may lead to synergistic cytotoxicity in MPN cells. We first treated 2 MPN cell lines harboring the JAK2 V617F mutation (SET2 and HEL) with veliparib at increasing concentrations and measured cell proliferation. SET2 and HEL cells were relatively sensitive to veliparib (IC 50 of 11.3 μM and 74.2 μM, respectively). We next treated cells with increasing doses of busulfan in combination with 4 μM veliparib and found that the busulfan IC 50 decreased from 27 μM to 4 μM in SET2 cells and from 45.1 μM to 28.1 μM in HEL cells. The mean combination index was.55 for SET2 cells and.40 for HEL cells. Combination treatment of SET2 cells caused G2M arrest in 53% of cells, compared with 30% with veliparib alone and 35% with busulfan alone. G2M arrest was associated with activation of the ATR-Chk1 pathway, as shown by an immunofluorescence assay for phosphorylated Chk1 (p-Chk1). We then tested in vivo the effect of combined low doses of busulfan and veliparib in a JAK2 V617F MPN-AML xenotransplant model. Vehicle- and veliparib-treated mice had similar median survival of 39 and 40 days, respectively. Combination treatment increased median survival from 47 days (busulfan alone) to 50 days (P =.02). Finally, we tested the combined effect of busulfan and veliparib on CD34 + cells obtained from the bone marrow or peripheral blood of 5 patients with JAK2 V617F -mutated and 2 patients with CALR-mutated MF. MF cells treated with the combination of veliparib and busulfan showed reduced colony formation compared with busulfan alone (87% versus 68%; P =.001). In contrast, treatment of normal CD34 + cells with veliparib did not affect colony growth. Here we show that in vivo confirmation that treatment with the PARP-1 inhibitor veliparib and busulfan results in synergistic cytotoxicity in MPN cells. Our data provide the rationale for testing novel pretransplantation conditioning regimens with combinations of PARP-1 inhibition and reduced doses of alkylators, such as busulfan and melphalan, for high-risk MPNs or MPN-derived acute myelogenous leukemia.

AB - Patients with high-risk myeloproliferative neoplasms (MPNs), and in particular myelofibrosis (MF), can be cured only with allogeneic hematopoietic stem cell transplantation (HSCT). Because MPNs and JAK2 V617F -mutated cells show genomic instability, stalled replication forks, and baseline DNA double-strand breaks, DNA repair inhibition with poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors represents a potential novel therapy. Because the alkylating agent busulfan is integral in conditioning regimens for HSCT and leads to stalled replication forks through DNA strand cross-linking, we hypothesized that PARP inhibition with veliparib in combination with busulfan may lead to synergistic cytotoxicity in MPN cells. We first treated 2 MPN cell lines harboring the JAK2 V617F mutation (SET2 and HEL) with veliparib at increasing concentrations and measured cell proliferation. SET2 and HEL cells were relatively sensitive to veliparib (IC 50 of 11.3 μM and 74.2 μM, respectively). We next treated cells with increasing doses of busulfan in combination with 4 μM veliparib and found that the busulfan IC 50 decreased from 27 μM to 4 μM in SET2 cells and from 45.1 μM to 28.1 μM in HEL cells. The mean combination index was.55 for SET2 cells and.40 for HEL cells. Combination treatment of SET2 cells caused G2M arrest in 53% of cells, compared with 30% with veliparib alone and 35% with busulfan alone. G2M arrest was associated with activation of the ATR-Chk1 pathway, as shown by an immunofluorescence assay for phosphorylated Chk1 (p-Chk1). We then tested in vivo the effect of combined low doses of busulfan and veliparib in a JAK2 V617F MPN-AML xenotransplant model. Vehicle- and veliparib-treated mice had similar median survival of 39 and 40 days, respectively. Combination treatment increased median survival from 47 days (busulfan alone) to 50 days (P =.02). Finally, we tested the combined effect of busulfan and veliparib on CD34 + cells obtained from the bone marrow or peripheral blood of 5 patients with JAK2 V617F -mutated and 2 patients with CALR-mutated MF. MF cells treated with the combination of veliparib and busulfan showed reduced colony formation compared with busulfan alone (87% versus 68%; P =.001). In contrast, treatment of normal CD34 + cells with veliparib did not affect colony growth. Here we show that in vivo confirmation that treatment with the PARP-1 inhibitor veliparib and busulfan results in synergistic cytotoxicity in MPN cells. Our data provide the rationale for testing novel pretransplantation conditioning regimens with combinations of PARP-1 inhibition and reduced doses of alkylators, such as busulfan and melphalan, for high-risk MPNs or MPN-derived acute myelogenous leukemia.

KW - Alkylator chemotherapy

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KW - PARP-1

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