Yasuhiko Kamikubo

Generation of induced pluripotent stem cells from primary chronic myelogenous leukemia patient samples.

Induced pluripotent stem cells (iPSCs) can be generated by the expression of defined transcription factors not only from normal tissue, but also from malignant cells. Cancer-derived iPSCs are expected to provide a novel experimental opportunity to establish the disease model. We generated iPSCs from imatinib-sensitive chronic myelogenous leukemia (CML) patient samples. Remarkably, the CML-iPSCs were resistant to imatinib although they consistently expressed BCR-ABL oncoprotein. In CML-iPSCs, the phosphorylation of ERK1/2, AKT, and JNK, which are essential for the maintenance of both BCR-ABL (+) leukemia cells and iPSCs, were unchanged after imatinib treatment, whereas the phosphorylation of signal transducer and activator of transcription (STAT)5 and CRKL was significantly decreased. These results suggest that the signaling for iPSCs maintenance compensates for the inhibition of BCR-ABL. CML-iPSC-derived hematopoietic cells recovered the sensitivity to imatinib although CD34(+)38(-)90(+)45(+) immature cells were resistant to imatinib, which recapitulated the pathophysiologic feature of the initial CML. CML-iPSCs provide us with a novel platform to investigate CML pathogenesis on the basis of patient-derived samples.

Accelerated leukemogenesis by truncated CBFβ-SMMHC defective in high-affinity binding with RUNX1

Dominant RUNX1 inhibition has been proposed as a common pathway for CBF leukemia. CBF beta-SMMHC, a fusion protein in human acute myeloid leukemia (AML), dominantly inhibits RUNX1 largely through its RUNX1 high-affinity binding domain (HABD). However, the type I CBF beta-SMMHC fusion in AML patients lacks HABD. Here, we report that the type I CBF beta-SMMHC protein binds RUNX1 inefficiently. Knockin mice expressing CBF beta-SMMHC with a HABD deletion developed leukemia quickly, even though hematopoietic defects associated with Runx1-inhibition were partially rescued. A larger pool of leukemia-initiating cells, increased MN1 expression, and retention of RUNX1 phosphorylation are potential mechanisms for accelerated leukemia development in these mice. Our data suggest that RUNX1 dominant inhibition may not be a critical step for leukemogenesis by CBF beta-SMMHC.

Inhibition of Cell Growth by Conditional Expression of kpm, a Human Homologue of Drosophila warts/lats Tumor Suppressor

kpm is a human serine/threonine kinase that is homologous to Drosophila tumor suppressor warts/lats and its mammalian homologue LATS1. In order to define the biological function of kpm, we generated stable transfectants of wild-type kpm (kpm-wt), a kinase-dead mutant ofkpm (kpm-kd), and luciferase in HeLa Tet-Off cells under the tetracycline-responsive promoter. Western blot analysis showed that high levels of expression of kpm-wt as well as kpm-kd with an apparent mass of 150 kDa were induced after the removal of doxycycline. Induction of kpm-wt expression resulted in a marked decline in viable cell number measured by both trypan blue dye exclusion and MTT assay, whereas that of kpm-kd or luciferase had no effect. We then analyzed the cell cycle progression and apoptosis upon induction of kpm expression. 2–3 days after removal of doxycycline, cells underwent G2/M arrest, demonstrated by flow cytometric analysis of propidium iodide incorporation and MPM-2 reactivity. In vitro kinase assay showed that induction of kpm-wt led to down-regulation of kinase activity of the Cdc2-cyclin B complex, which was accompanied by an increase in the hyperphosphorylated form of Cdc2 and a change of phosphorylation status of Cdc25C. Furthermore, both DAPI staining and TUNEL assay showed that the proportion of apoptotic cells increased as kpm expression was induced. Taken together, these results indicate that kpm negatively regulates cell growth by inducing G2/M arrest and apoptotic cell death through its kinase activity.

Molecular cloning of a novel human protein kinase, kpm, that is homologous to warts/lats, a Drosophila tumor suppressor

A novel human protein kinase, designated kpm, was identified and molecularly cloned. The isolated cDNA clone had an open reading frame consisting of 1088 amino acid residues with a putative kinase domain located near the carboxy-terminus. Homology search revealed that kpm belongs to a subfamily of serine/threonine protein kinases including warts/lats, a Drosophila tumor suppressor. Among these, kpm is most homologous to, but distinct from, recently reported LATS1, a human homolog of Drosophila warts/lats. Northern blot analysis disclosed that kpm is expressed as a 6.0 kb transcript in most of the tissues examined and also as an additional shorter 4.0 kb transcript in testis. Western blotting using polyclonal rabbit anti-kpm antibody detected kpm protein as a band with an apparent Mr of 150 kD. Immune complex kinase assay of HA-tagged kpm showed that kpm had kinase activity and phosphorylated itself in vitro. Studies with synchronized HeLa cells indicated that kpm protein was expressed relatively constantly throughout the cell cycle and underwent significant phosphorylation at mitotic phase. These results suggest that kpm plays a role in cell cycle progression during mitosis and its deletion or dysfunction might be involved in certain types of human cancers.

Genetic regulation of the RUNX transcription factor family has antitumor effects

Runt-related transcription factor 1 (RUNX1) is generally considered to function as a tumor suppressor in the development of leukemia, but a growing body of evidence suggests that it has pro-oncogenic properties in acute myeloid leukemia (AML). Here we have demonstrated that the antileukemic effect mediated by RUNX1 depletion is highly dependent on a functional p53-mediated cell death pathway. Increased expression of other RUNX family members, including RUNX2 and RUNX3, compensated for the antitumor effect elicited by RUNX1 silencing, and simultaneous attenuation of all RUNX family members as a cluster led to a much stronger antitumor effect relative to suppression of individual RUNX members. Switching off the RUNX cluster using alkylating agent–conjugated pyrrole-imidazole (PI) polyamides, which were designed to specifically bind to consensus RUNX-binding sequences, was highly effective against AML cells and against several poor-prognosis solid tumors in a xenograft mouse model of AML without notable adverse events. Taken together, these results identify a crucial role for the RUNX cluster in the maintenance and progression of cancer cells and suggest that modulation of the RUNX cluster using the PI polyamide gene-switch technology is a potential strategy to control malignancies.

Role of RUNX1 in hematological malignancies

RUNX1 is a member of the core-binding factor family of transcription factors and is indispensable for the establishment of definitive hematopoiesis in vertebrates. RUNX1 is one of the most frequently mutated genes in a variety of hematological malignancies. Germ line mutations in RUNX1 cause familial platelet disorder with associated myeloid malignancies. Somatic mutations and chromosomal rearrangements involving RUNX1 are frequently observed in myelodysplastic syndrome and leukemias of myeloid and lymphoid lineages, that is, acute myeloid leukemia, acute lymphoblastic leukemia, and chronic myelomonocytic leukemia. More recent studies suggest that the wild-type RUNX1 is required for growth and survival of certain types of leukemia cells. The purpose of this review is to discuss the current status of our understanding about the role of RUNX1 in hematological malignancies.

KIT with D816 mutations cooperates with CBFB-MYH11 for leukemogenesis in mice

KIT mutations are the most common secondary mutations in inv(16) acute myeloid leukemia (AML) patients and are associated with poor prognosis. It is therefore important to verify that KIT mutations cooperate with CBFB-MYH11, the fusion gene generated by inv(16), for leukemogenesis. Here, we transduced wild-type and conditional Cbfb-MYH11 knockin (KI) mouse bone marrow (BM) cells with KIT D816V/Y mutations. KIT transduction caused massive BM Lin(-) cell death and fewer colonies in culture that were less severe in the KI cells. D816Y KIT but not wild-type KIT enhanced proliferation in Lin(-) cells and led to more mixed lineage colonies from transduced KI BM cells. Importantly, 60% and 80% of mice transplanted with KI BM cells expressing D816V or D816Y KIT, respectively, died from leukemia within 9 months, whereas no control mice died. Results from limiting dilution transplantations indicate higher frequencies of leukemia-initiating cells in the leukemia expressing mutated KIT. Signaling pathway analysis revealed that p44/42 MAPK and Stat3, but not AKT and Stat5, were strongly phosphorylated in the leukemia cells. Finally, leukemia cells carrying KIT D816 mutations were sensitive to the kinase inhibitor PKC412. Our data provide clear evidence for cooperation between mutated KIT and CBFB-MYH11 during leukemogenesis.

Loss of AML1/Runx1 accelerates the development of MLL-ENL leukemia through down-regulation of p19ARF.

Dysfunction of AML1/Runx1, a transcription factor, plays a crucial role in the development of many types of leukemia. Additional events are often required for AML1 dysfunction to induce full-blown leukemia; however, a mechanistic basis of their cooperation is still elusive. Here, we investigated the effect of AML1 deficiency on the development of MLL-ENL leukemia in mice. Aml1 excised bone marrow cells lead to MLL-ENL leukemia with shorter duration than Aml1 intact cells in vivo. Although the number of MLL-ENL leukemia-initiating cells is not affected by loss of AML1, the proliferation of leukemic cells is enhanced in Aml1-excised MLL-ENL leukemic mice. We found that the enhanced proliferation is the result of repression of p19(ARF) that is directly regulated by AML1 in MLL-ENL leukemic cells. We also found that down-regulation of p19(ARF) induces the accelerated onset of MLL-ENL leukemia, suggesting that p19(ARF) is a major target of AML1 in MLL-ENL leukemia. These results provide a new insight into a role for AML1 in the progression of leukemia.

Cbfb/Runx1-repression independent blockage of differentiation and accumulation of Csf2rb expressing cells by Cbfb-MYH11

It is known that CBFB-MYH11, the fusion gene generated by inversion of chromosome 16 in human acute myeloid leukemia, is causative for oncogenic transformation. However, the mechanism by which CBFB-MYH11 initiates leukemogenesis is not clear. Previously published reports showed that CBFB-MYH11 dominantly inhibits RUNX1 and CBFB, and such inhibition has been suggested as the mechanism for leukemogenesis. Here we show that Cbfb-MYH11 caused Cbfb/Runx1 repression-independent defects in both primitive and definitive hematopoiesis. During primitive hematopoiesis, Cbfb-MYH11 delayed differentiation characterized by sustained expression of Gata2, Il1rl1, and Csf2rb, a phenotype not found in Cbfb and Runx1 knockout mice. Expression of Cbfb-MYH11 in the bone marrow induced the accumulation of abnormal progenitor-like cells expressing Csf2rb in preleukemic mice. The expression of all 3 genes was detected in most human and murine CBFB-MYH11(+) leukemia samples. Interestingly, Cbfb-MYH11(+) preleukemic progenitors and leukemia-initiating cells did not express Csf2rb, although the majority of leukemia cells in our Cbfb-MYH11 knockin mice were Csf2rb(+). Therefore Csf2rb can be used as a negative selection marker to enrich preleukemic progenitor cells and leukemia-initiating cells from Cbfb-MYH11 mice. These results suggest that Cbfb/Runx1 repression-independent activities contribute to leukemogenesis by Cbfb-MYH11.

Adverse prognostic impact of KIT mutations in childhood CBF-AML: the results of the Japanese Pediatric Leukemia/Lymphoma Study Group AML-05 trial

Adverse prognostic impact of KIT mutations in childhood CBF-AML: the results of the Japanese Pediatric Leukemia/Lymphoma Study Group AML-05 trial