Disease recurrence is the major problem in the treatment of acute myeloid leukemia (AML). Relapse is driven by leukemia stem cells, a chemoresistant subpopulation capable of re-establishing disease. Patients with p53 mutant AML are at an extremely high risk of relapse. B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is required for the self-renewal and maintenance of AML stem cells. Here we studied the effects of a novel small molecule inhibitor of BMI-1, PTC596, in AML cells. Treatment with PTC596 reduced MCL-1 expression and triggered several molecular events consistent with induction of mitochondrial apoptosis: loss of mitochondrial membrane potential, BAX conformational change, caspase-3 cleavage and phosphatidylserine externalization. PTC596 induced apoptosis in a p53-independent manner. PTC596 induced apoptosis along with the reduction of MCL-1 and phosphorylated AKT in patient-derived CD34+CD38low/− stem/progenitor cells. Mouse xenograft models demonstrated in vivo anti-leukemia activity of PTC596, which inhibited leukemia cell growth in vivo while sparing normal hematopoietic cells. Our results indicate that PTC596 deserves further evaluation in clinical trials for refractory or relapsed AML patients, especially for those with unfavorable complex karyotype or therapy-related AML that are frequently associated with p53 mutations.

Curing patients with acute myeloid leukemia (AML) remains a therapeutic challenge. The polycomb complex protein B‐cell‐specific Moloney murine leukemia virus integration site 1 (BMI‐1) is required for the self‐renewal and maintenance of leukemia stem cells. We investigated the prognostic significance of BMI‐1 in AML and the effects of a novel small molecule selective inhibitor of BMI‐1, PTC‐209. BMI‐1 protein expression was determined in 511 newly diagnosed AML patients together with 207 other proteins using reverse‐phase protein array technology. Patients with unfavorable cytogenetics according to Southwest Oncology Group criteria had higher levels of BMI‐1 compared to those with favorable (P = 0.0006) or intermediate cytogenetics (P = 0.0061), and patients with higher levels of BMI‐1 had worse overall survival (55.3 weeks vs. 42.8 weeks, P = 0.046). Treatment with PTC‐209 reduced protein level of BMI‐1 and its downstream target mono‐ubiquitinated histone H2A and triggered several molecular events consistent with the induction of apoptosis, this is, loss of mitochondrial membrane potential, caspase‐3 cleavage, BAX activation, and phosphatidylserine externalization. PTC‐209 induced apoptosis in patient‐derived CD34+ CD38low/− AML cells and, less prominently, in CD34− differentiated AML cells. BMI‐1 reduction by PTC‐209 directly correlated with apoptosis induction in CD34+ primary AML cells (r = 0.71, P = 0.022). However, basal BMI‐1 expression was not a determinant of AML sensitivity. BMI‐1 inhibition, which targets a primitive AML cell population, might offer a novel therapeutic strategy for AML.

Despite the development of the novel Bruton tyrosine kinase inhibitor ibrutinib, mantle cell lymphoma (MCL) remains an incurable B-cell non-Hodgkin lymphoma. BMI-1 is required for the self-renewal and maintenance of MCL-initiating stem cells. Upregulation of BMI-1 has been reported in MCL patients, especially in those with refractory/relapsed disease. We studied the effects of a novel small-molecule selective inhibitor of BMI1 expression, PTC596, in MCL cells. Eight MCL cell lines and patient-derived samples were exposed to PTC596. PTC596 induced mitochondrial apoptosis, as evidenced by loss of mitochondrial membrane potential, caspase-3 cleavage, BAX activation, and phosphatidylserine externalization. There was a positive correlation between baseline BMI-1 protein levels and PTC596-induced apoptosis. p53 status did not affect sensitivity to PTC596. PTC596 effectively decreased BMI-1-expressing and tumor-initiating side population MCL cells (IC50: 138 nM) compared with ibrutinib, which modestly decreased side population cells. Interestingly, PTC596, reported to target cancer stem cells, decreased MCL-1 expression levels and antagonized ibrutinib-induced increase in MCL-1 expression, leading to synergistic apoptosis induction in MCL cells. There are currently no drugs that specifically target cancer stem cell fractions, and a reduction in BMI-1 protein by PTC596 may offer a novel therapeutic strategy for MCL.

PPM1D is a serine/threonine phosphatase that negatively regulates key DNA damage response proteins, such as p53, p38 MAPK, histone H2A.X, and ATM. We investigated the pathophysiological significance of PPM1D and its therapeutic targeting by the novel PPM1D inhibitor GSK2830371 in mantle cell lymphoma (MCL). Oncomine-based analyses indicated increased PPM1D mRNA levels in MCL cells compared with their normal counterpart cells. Higher PPM1D expression was associated with higher expression of the proliferation gene signature and poorer prognosis in patients. Eight MCL (three p53 wild-type and five mutant) cell lines were exposed to GSK2830371. GSK2830371 inhibited the cell growth, being prominent in p53 wild-type cells. GSK2830371 induced apoptosis in sensitive cells, as evidenced by induction of phosphatidylserine externalization and loss of mitochondrial membrane potential. p53 knockdown de-sensitized cell sensitivity. GSK2830371 increased the levels of total and Ser15-phosphorylated p53, and p53 targets p21 and PUMA. GSK2830371 and the MDM2 inhibitor Nutlin-3a acted synergistically in p53 wild-type cells. Interestingly, GSK2830371 sensitized MCL cells to bortezomib and doxorubicin in p53 wild-type and mutant cells; p38 signaling appeared to be involved in the GSK2830371/bortezomib lethality. PPM1D inhibition may represent a novel therapeutic strategy for MCL, which can be exploited in combination therapeutic strategies for MCL.

A minor fraction of leukemia cells, leukemia stem cells, have been shown to be highly resistant to current therapies and thought to be responsible for recurrence. BMI-1, a part of polycomb repressive complex 1 (PRC1) is essential for the self-renewal of normal hematopoietic and leukemia stem cells. PTC-209 is a novel selective transcriptional inhibitor of BMI-1, which has been shown to have antitumor activity against cancer-initiating cells in colorectal cancer. We investigated the prognostic significance of BMI-1 in acute myeloid leukemia (AML) using reversed phase protein array and effects of the BMI-1 inhibitor PTC-209 on primary and leukemia cell lines. BMI-1 protein expression was determined in bulk AML blasts from 511 newly diagnosed patients. BMI-1 expression was higher in unfavorable cytogenetics (n=252, median 0.068) compared to intermediate (n=225, median -0.116, P = 0.017) or favorable cytogenetics (n=34, median -0.338, P = 0.0007 versus unfavorable, 0.05 versus intermediate). Higher BMI-1 levels were associated with shorter median overall survival (42.8 versus 55.3 weeks, P = 0.046 Log Rank test). There was no correlation between BMI-1 levels and percentages of CD34 -positive cells (r = 0.07). A total of 6 AML (MOLM-13, OCI-AML3, MV4-11, NB4, HL60 and U-937) and 5 ALL (Reh, NALM6, Jurkat, Raji and MOLT-4) cell lines were exposed to PTC-209 for 48 hours. PTC-209 exhibited dose- and time-dependent anti-proliferative and cytotoxic activities. The IC50 values (concentration at which cell growth is inhibited by 50% at 48 hours of exposure) were 0.33 ± 0.04 µM (mean ± SEM) for AML and 0.55 ± 0.09 µM for ALL, indicating potent anti-proliferative effects. In contrast, PTC-209 showed differential cytotoxic effects between AML and ALL cells. The ED50 values (effective concentration inducing 50% killing as measured by Annexin V positivity) were no more than 2.5 µM in 5 of 6 AML lines while they were higher than 10 µM in 3 out of 5 ALL cell lines, implicating that BMI-1 is more critical in AML than ALL. Treatment with PTC-209 triggered several molecular events consistent with induction of apoptosis in sensitive lines (e.g. MV4-11 and MOLM-13): conformational change of BAX (i.e., BAX activation), loss of mitochondrial membrane potential (MMP), caspase-3 activation and DNA fragmentation in addition to phosphatidylserine (PS) externalization. Eighteen-hour treatment of MV4-11 cells with 2.5 µM PTC-209 led to compound-specific induction of conformationally active BAX (31%), MMP loss (80%) and caspase-3 cleavage (38%). qRT-PCR showed reduced transcript level of BMI-1 (61% reduction) after 6-hour PTC-209 exposure in MV4-11 cells. PTC-209 induced PS externalization in primary AML cells (82.5 ± 4.3% after 48-hour treatment with 2 µM PTC-209, n = 6) and to a lesser degree, in ALL cells (33.7 ± 13.4%, n = 4, p < 0.05). Importantly, CD34+CD38– AML progenitor cells were as sensitive to PTC-209 as C34– more mature AML cells. Normal lymphocytes were resistant to PTC-209 (9.1 ± 4.6% even at 10 µM). Collectively, BMI-1 inhibition by small molecule inhibitors could be developed into a novel therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

Leukemia stem cells are resistant to chemotherapeutic agents and contribute to disease relapse. BMI-1, a part of polycomb repressive complex 1 (PRC1) is essential for the self-renewal of normal hematopoietic and leukemia stem cells. We have reported that acute myeloid leukemia (AML) patients with higher levels of BMI-1 have worse overall survival and that the translational BMI-1 inhibitor PTC-209 induces apoptosis in patient-derived CD34+CD38low/- AML cells. PTC596 is a second-generation BMI-1 inhibitor that accelerates BMI-1 degradation. Based on preclinical data showing excellent anti-tumor activities against solid cancers, PTC596 is currently in PH1 clinical development. We here investigated preclinical activities of PTC596 against acute leukemias in vitro and in vivo. A total of 6 AML (MOLM-13, OCI-AML3, U-937, MV4-11, MOLM-14 and HL60) and 3 acute lymphoblastic leukemia (ALL) (Reh, NALM6 and MOLT-4) cell lines were exposed to PTC596 for 48 hours. PTC596 exhibited dose- and time-dependent anti-proliferative and cytotoxic (apoptotic) activities. The IC50 values (concentration at which cell growth is inhibited by 50% at 48 hours of exposure) were 30.7 ± 4.1 nM (mean ± SEM) for AML and 37.7 ± 8.0 nM for ALL. The ED50 values (effective concentration inducing 50% killing as measured by Annexin V/PI induction) were 60.3 ± 6.7 nM for AML and 77.8 ± 11.5 nM for ALL. PTC596 induced apoptosis in AML cells irrespective of their p53 status. 72-hour treatment of MOLM-13 and OCI-AML3 cells with 100nM PTC596 reduced total BMI-1 protein levels by 87% and 61%, respectively. Forced overexpression of BMI-1 in K562 cells abrogated PTC596-induced apoptosis (∼ 80% at 50 nM PTC596; P &lt; 0.05). 16-hour treatment of MOLM-13 cells with 200nM PTC596 resulted in reduced levels of non-phosphorylated BMI-1 by 85%, along with increased levels of phosphorylated BMI-1. PTC596 reduced the levels of ubiquitylated histone H2A, which indicates a deactivation of the PRC1 complex function. The anti-leukemia activity of PTC596 was tested in xenograft (K562) and syngeneic (L210) mouse models. PTC596 at a daily dose of 20 mg/kg significantly delayed an enlargement of the implanted tumor of K562 by ∼55%. PTC596 significantly prolonged survival of mice injected with syngeneic L210 leukemia cells (27 versus 37 days; 76% extension of lifespan with a median survival time; P &lt; 0.05). Collectively, BMI-1 inhibition by PTC596 offers a novel therapeutic strategy for acute leukemias. Citation Format: Yuki Nishida, Aya Maeda, Liangxian Cao, Melissa Dumble, Shinya Kimura, Thomas W. Davis, Kensuke Kojima. Targeting BMI-1 by the novel BMI-1 inhibitor PTC596 in acute leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A130.

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Introduction Mantle cell lymphoma (MCL) is a distinct type of non-Hodgkin lymphoma genetically characterized by balanced t(11:14) translocation and cyclin D1 overexpression. A great majority of patients with MCL remain incurable. The selection of chemoresistant subclones occurs during therapies, leading to relapse of MCL. Many chemotherapeutic agents induce DNA damage and activate the tumor suppressor p53. Recently, non-genotoxic p53 activators like MDM2 (master regulator of p53) inhibitors and XPO1 (nuclear exporter for p53) inhibitors have entered clinical trials. p53 stabilization, however, has not always led to p53-mediated transcriptional activation in MCL. PPM1D is a serine/threonine phosphatase that negatively regulates key DNA damage response proteins, such as p53, p38 MAPK, histone H2A.X, and ATM. PPM1D has been thought to be an oncoprotein that inhibits p53. PPM1D overexpression or amplification has been reported in various cancers, including lung, breast, kidney and ovarian cancers. We investigated the pathophysiological significance of PPM1D and its therapeutic targeting by the novel PPM1D inhibitor GSK2830371 (GSK) (Nat Chem Biol 2014) in MCL. RESULTS Oncomine-based analyses indicated increased PPM1D mRNA levels in MCL cells compared with their normal counterpart B-lymphocytes. PPM1D mRNA levels positively correlated with CCND1 (Cyclin D1) mRNA levels (r = 0.33, P = 0.0014; n = 92) and with proliferation signature averages (r = 0.54, P < 0.0001; n = 92) in a series of MCL samples. Increased PPM1D expression at diagnosis was itself associated with a poorer prognosis in MCL patients (median overall survival of 3.9 years and 1.4 years for cases in the lowest and highest PPM1D expression tertiles, respectively; P = 0.0047). PPM1D levels in MCL were as high as those in aggressive lymphomas including Burkitt's lymphoma and diffuse large B-cell lymphoma, and were significantly higher than those in indolent lymphomas including chronic lymphocytic leukemia/small lymphocytic lymphoma (P = 0.0076) and follicular lymphoma (P = 0.011). Eight MCL (three p53 wild-type (WT) and five mutant (MUT)) cell lines were exposed to GSK. GSK inhibited the cell growth, being more prominent in p53 WT cells (48.3 ± 9.8% versus 14.8 ± 4.7% growth inhibition, P = 0.036). In sensitive cells, GSK caused a significant loss of mitochondrial membrane potential in addition to increased annexin V positivity, indicating apoptosis induction. Stable p53-specific shRNA-expressing cells were generated in p53 WT Z-138 and JVM-2 cells, with > 85% knockdown efficiency. p53 knockdown cells were less susceptible to GSK than control cells, indicating that GSK utilizes p53-mediated signaling to eradicate MCL cells. This idea is supported by the Western blot data that GSK treatment increased total and phosphorylated p53 levels and those of p53 targets p21 and PUMA. Basal and GSK-induced levels of PPM1D and its target proteins NFkB-p65, p38 MAPK, and histone H2A.X did not predict MCL cell sensitivity to GSK. GSK and the MDM2 inhibitor Nutlin-3a acted synergistically in p53 WT MCL cells. Importantly, GSK sensitized MCL cells to bortezomib (BTZ) and doxorubicin (DOX) irrespective of p53 mutational status. BTZ and DOX are clinically active agents against MCL. In p53 WT cells, GSK/BTZ and GSK/DOX combinations potently activated p53-mediated apoptosis signaling. In p53 MUT cells, combination treatment did not activate p53 signaling. Instead, p38 signaling appeared to be actively involved in the GSK/BTZ lethality, as the selective p38 inhibitor SB203580 significantly attenuated bortezomib- and GSK/bortezomib-induced lethality. Activation of p38 MAPK has been found to cause MCL cell death. SB203580 did not protect MCL cell from doxorubicin- or GSK/doxorubicin-induced lethality. CONCLUSION PPM1D inhibition increases levels of phosphorylated p53 at Ser15 in a non-genotoxic manner, which enhances p53-mediated transcription in MCL cells. In addition, PPM1D inhibition shows anti-lymphoma effects in p53 MUT cells, partially through activation of p38 signaling. Although a single-agent activity of GSK was modest against MCL cells, especially against those with mutant p53, its p53-independent potentiation effect on BTZ and DOX may support the use of PPM1D inhibitors as part of a combination therapeutic strategy for MCL. Collectively, PPM1D inhibition may offer a novel therapeutic strategy for MCL. Disclosures No relevant conflicts of interest to declare.