Seiji Kakiuchi

Small-molecule Hedgehog inhibitor attenuates the leukemia-initiation potential of acute myeloid leukemia cells.

Aberrant activation of the Hedgehog signaling pathway has been implicated in the maintenance of leukemia stem cell populations in several model systems. PF‐04449913 (PF‐913) is a selective, small‐molecule inhibitor of Smoothened, a membrane protein that regulates the Hedgehog pathway. However, details of the proof‐of‐concept and mechanism of action of PF‐913 following administration to patients with acute myeloid leukemia (AML) are unclear. This study examined the role of the Hedgehog signaling pathway in AML cells, and evaluated the in vitro and in vivo effects of the Smoothened inhibitor PF‐913. In primary AML cells, activation of the Hedgehog signaling pathway was more pronounced in CD34+ cells than CD34− cells. In vitro treatment with PF‐913 induced a decrease in the quiescent cell population accompanied by minimal cell death. In vivo treatment with PF‐913 attenuated the leukemia‐initiation potential of AML cells in a serial transplantation mouse model, while limiting reduction of tumor burden in a primary xenotransplant system. Comprehensive gene set enrichment analysis revealed that PF‐913 modulated self‐renewal signatures and cell cycle progression. Furthermore, PF‐913 sensitized AML cells to cytosine arabinoside, and abrogated resistance to cytosine arabinoside in AML cells cocultured with HS‐5 stromal cells. These findings imply that pharmacologic inhibition of Hedgehog signaling attenuates the leukemia‐initiation potential, and also enhanced AML therapy by sensitizing dormant leukemia stem cells to chemotherapy and overcoming resistance in the bone marrow microenvironment.

NANOG Expression as a Responsive Biomarker during Treatment with Hedgehog Signal Inhibitor in Acute Myeloid Leukemia

Aberrant activation of the Hedgehog (Hh) signaling pathway is involved in the maintenance of leukemic stem cell (LSCs) populations. PF-0444913 (PF-913) is a novel inhibitor that selectively targets Smoothened (SMO), which regulates the Hh pathway. Treatment with PF-913 has shown promising results in an early phase study of acute myeloid leukemia (AML). However, a detailed mode of action for PF-913 and relevant biomarkers remain to be elucidated. In this study, we examined bone marrow samples derived from AML patients under PF-913 monotherapy. Gene set enrichment analysis (GSEA) revealed that PF-913 treatment affected the self-renewal signature and cell-cycle regulation associated with LSC-like properties. We then focused on the expression of a pluripotency factor, NANOG, because previous reports showed that a downstream effector in the Hh pathway, GLI, directly binds to the NANOG promoter and that the GLI-NANOG axis promotes stemness and growth in several cancers. In this study, we found that a change in NANOG transcripts was closely associated with GLI-target genes and NANOG transcripts can be a responsive biomarker during PF-913 therapy. Additionally, the treatment of AML with PF-913 holds promise, possibly through inducing quiescent leukemia stem cells toward cell cycling.

An mTORC1/2 kinase inhibitor enhances the cytotoxicity of gemtuzumab ozogamicin by activation of lysosomal function

Gemtuzumab ozogamicin (GO), the first antibody-drug conjugate (ADC), has attracted the interest of hematologists because more than 90% of acute myeloid leukemia (AML) blasts express its target, CD33. Although GO and subsequently developed ADCs depend on lysosomes for activation, lysosome number and activity in tumor cells has not been well elucidated. In this study, we investigated whether an mTORC1/2 kinase inhibitor, PP242, which was reported to activate lysosomal function, potentiates the cytotoxicity of GO in AML cells. Eight AML cell lines (U937, THP-1, SKM-1, SKK-1, SKNO-1, HL-60, MARIMO and KO52) were treated with GO and PP242. The cytotoxic effect of GO was enhanced by concurrent treatment with a non-cytotoxic concentration (500 nM) of PP242 in most cell lines, except MARIMO and KO52 cells. We then used LysoTracker to label acidic lysosomes in U937, THP-1, SKM-1, MARIMO and KO52 cells. LysoTracker fluorescence was dramatically increased by treatment with PP242 in U937, THP-1 and SKM-1 cells, and the intensified fluorescence was retained with PP242 + GO. In contrast, PP242 did not induce a significant increase in fluorescence in MARIMO cells, consistent with the lack of combinatory cytotoxicity. LysoTracker fluorescence was also increased by PP242 in KO52 cells, which have been reported to strongly express multidrug resistance (MDR). Further, PP242 suppressed GO-induced Chk1 activation and G2/M cell cycle arrest, which in turn triggered cell cycle promotion and cell death. These results indicate that inhibition of mTORC1/2 kinase by PP242 enhanced the cytotoxicity of GO by increasing lysosomal compartments and promoting the cell cycle via suppression of GO-induced Chk1 activation. This combination may represent an attractive new therapeutic strategy for the treatment of leukemia.

Expression of a novel ZMYND11/MBTD1 fusion transcript in CD7+CD56+ acute myeloid leukemia with t(10;17)(p15;q21)

The t(10;17)(p15;q21) translocation is a very rare but recurrent cytogenetic aberration in acute myeloid leukemia (AML), which would be classified as AML, not otherwise specified by World Health Organization (WHO), generally with classification as M0 or M1 by French–American–British (FAB) criteria [1–8]. Recently, co-localization between the ZMYND11 (zinc finger MYND-type containing 11, alias BS69) gene on 10p15.3 and the MBTD1 (mbt domain containing 1) gene on 17q21.33 was shown by fluorescence in situ hybridization (FISH) in two cases of AML M1 with t(10;17)(p15;q21) [7]. Furthermore, molecular analyses identified a ZMYND11/MBTD1 fusion transcript in a recent case of AML M0 with t(10;17) [8]. Nucleotide sequencing revealed an in-frame fusion of ZMYND11 exon 12 to MBTD1 exon 3 generated on der(17)t(10;17). The resultant ZMYND11/MBTD1 fusion protein was supposed to contribute to HOXA overexpression, which is a leukemogenic pathway to AML, whereas the reciprocal MBTD1/ZMYND11 fusion was predicted to lack a productive start codon [8]. Here, we describe an unusual case of AML with t(10;17)(p15;q21), demonstrating a novel ZMYND11/MBTD1 fusion transcript and a CD7þCD56þ immunophenotype. A 67-year-old man was admitted to our hospital because of the appearance of blasts in his peripheral blood. He did not show any extramedullary mass, including lymphadenopathy. The peripheral blood counts 6 months before were normal: hemoglobin 147 g/L, platelets 190 10/L, and leukocytes 4.2 10/L with no blasts. Peripheral blood values on admission were hemoglobin 126 g/L, platelets 199 10/L, and leukocytes 3.8 10/L with 31% segmented neutrophils, 4% monocytes, 45% lymphocytes, and 20% blasts. His bone marrow was normocellular with 18.2% blasts, 2.6% myelocytes, 3.8% metamyelocytes, 1.0% band forms, 20.8% segmented neutrophils, 3.2% monocytes, 22.4% lymphocytes, and 25.0% erythroblasts. Dysplastic changes of bone marrow cells were not apparent. Mediumto large-sized blasts showed fine nuclear chromatin, prominent nucleoli, a pale cytoplasm, and a lack of azurophilic granules (Figure 1(A)). These blasts were negative for myeloperoxidase (MPO) and periodic acid–Schiff staining (Figure 1(B)). Immunophenotyping by three-color flow cytometry using CD45/side scatter gating revealed that gated cells (21.0% of all bone marrow cells) were positive (>20%) for CD7 (94.2%), CD13 (39.0%), CD33 (97.7%), CD34 (91.3%), CD41 (60.7%), CD56 (94.4%), and HLA-DR (40.9%), but negative for other lymphoid markers, including CD3 (0.8%; Figure 1(C)). A diagnosis of de novo AML M0, or AML with minimal differentiation, was made. The patient received induction therapy with idarubicin and cytarabine according to a JALSG-AML201 protocol and achieved complete remission (CR). He then received a further four courses of conventional consolidation therapy, and has remained in hematological and cytogenetic CR for more than 14 months. Chromosome analysis of bone marrow cells on admission showed 47,XY,þY,t(10;17)(p15;q21)[8]/47,sl,del(12)(p?) [8]/46,XY[4] (Figure 1(D)). At hematological CR after induction therapy, the karyotype returned to 46,XY[20]. Spectral karyotyping (SKY) confirmed der(10)t(10;17)(p15;q21), whereas the small segment, 10p15!10pter, on der(17)t(10;17)(p15;q21) could not be visualized (Figure 1(E)). This segment may be smaller than the minimum genomic alteration detectable by SKY [9]. FISH with a PML/RARA probe revealed that the RARA signal at 17q21 remained on the der(17)t(10;17), indicating that the 17q21 breakpoint of t(10;17) was telomeric to RARA, as previously reported (data not shown) [6]. We next performed reverse transcription–polymerase chain reaction (RT–PCR) for the possible detection of ZMYND11/MBTD1 fusion transcripts. We designed a forward primer, ZMYND11-F (from ZMYND11 exon 11, 50GAGGACCGAGGTGAGGAAGA-30, cDNA positions 1443–1462 according to NCBI reference sequence NM_006624.5), and a reverse primer, MBTD1-R (from MBTD1 exon 3, 50-

A Case of Classical Hodgkin Lymphoma with Total Lymph Node Infarction

Lymph node infarction is very rare, and is frequently associated with neoplasms, such as malignant lymphoma and non-neoplastic disease, or interventions such as fine-needle aspiration (FNA). A 76-year-old-man presented with cervical lymph node swelling. Although FNA was performed, the findings were insufficient for a definitive diagnosis. Consequently, surgical biopsy of the cervical lymph node was performed, which revealed total infarction; a diagnosis of classical Hodgkin lymphoma was made later. Both lymphoma itself and FNA may cause total lymph node infarction, which makes diagnosis confusing. Therefore, it is important to repeat the biopsy rather than repeat FNA to correctly diagnose malignant lymphoma, including Hodgkin lymphoma.

Invasive Scopulariopsis alboflavescens infection in patient with acute myeloid leukemia

Scopulariopsis alboflavescens is a soil saprophyte that is widely distributed in nature. Recently, there have been increasing number of reports of invasive infections with Scopulariopsis species in immunocompromised patients. In this report, we described an adult woman with acute myeloid leukemia and who developed S. alboflavescens pneumonia. Liposomal amphotericin B and voriconazole combination therapy was unsuccessful and the patient died because of pneumonia. Scopulariopsis is highly resistant to available antifungal agents and almost invariably fatal. This case report should alert clinicians to the importance of listing Scopulariopsis as a pathogenic fungus in immunocompromised patients.

Rhabdomyolysis Caused by Candida parapsilosis in a Patient with Acute Myeloid Leukemia after Bone Marrow Transplantation

Rhabdomyolysis is characterized by a marked elevation of the creatine kinase (CK) levels and myoglobinuria, thus leading to renal dysfunction. Various viruses or bacteria can be etiologic agents, but mycosis has only rarely been reported to be a cause of rhabdomyolysis. In this report, we describe an adolescent male with acute myeloid leukemia who underwent allogeneic bone marrow transplantation and thereafter developed rhabdomyolysis and Candida parapsilosis fungemia almost at the same time. Following treatment for C. parapsilosis, the transaminase and CK levels both satisfactorily decreased. This case illustrates that C. parapsilosis infection may be a causative agent of rhabdomyolysis in immunocompromised patients.