Hiroko Oshiro

Clofarabine exerts antileukemic activity against cytarabine-resistant B-cell precursor acute lymphoblastic leukemia with low deoxycytidine kinase expression

Cytosine arabinoside (Ara‐C) is one of the key drugs for the treatment of acute myeloid leukemia. It is also used for consolidation therapy of acute lymphoblastic leukemia (ALL). Ara‐C is a deoxyadenosine analog and is phosphorylated to form cytosine arabinoside triphosphate (Ara‐CTP) as an active form. In the first step of the metabolic pathway, Ara‐C is phosphorylated to Ara‐CMP by deoxycytidine kinase (DCK). However, the current cumulative evidence in the association of the Ara‐C sensitivity in ALL appears inconclusive. We analyzed various cell lines for the possible involvement of DCK in the sensitivities of B‐cell precursor ALL (BCP‐ALL) to Ara‐C. Higher DCK expression was associated with higher Ara‐C sensitivity. DCK knockout by genome editing with a CRISPR‐Cas9 system in an Ara‐C‐sensitive‐ALL cell line induced marked resistance to Ara‐C, but not to vincristine and daunorubicin, indicating the involvement of DCK expression in the Ara‐C sensitivity of BCP‐ALL. DCK gene silencing due to the hypermethylation of a CpG island and reduced DCK activity due to a nonsynonymous variant allele were not associated with Ara‐C sensitivity. Clofarabine is a second‐generation deoxyadenosine analog rationally synthesized to improve stability and reduce toxicity. The IC50 of clofarabine in 79 BCP‐ALL cell lines was approximately 20 times lower than that of Ara‐C. In contrast to Ara‐C, although the knockout of DCK induced marked resistance to clofarabine, sensitivity to clofarabine was only marginally associated with DCK gene expression level, suggesting a possible efficacy of clofarabine for BCP‐ALL that shows relative Ara‐C resistance due to low DCK expression.

Anti-leukemic activity of bortezomib and carfilzomib on B-cell precursor ALL cell lines

Prognosis of childhood acute lymphoblastic leukemia (ALL) has been dramatically improved. However, prognosis of the cases refractory to primary therapy is still poor. Recent phase 2 study on the efficacy of combination chemotherapy with bortezomib (BTZ), a proteasome inhibitor, for refractory childhood ALL demonstrated favorable clinical outcomes. However, septic death was observed in over 10% of patients, indicating the necessity of biomarkers that could predict BTZ sensitivity. We investigated in vitro BTZ sensitivity in a large panel of ALL cell lines that acted as a model system for refractory ALL, and found that Philadelphia chromosome-positive (Ph+) ALL, IKZF1 deletion, and biallelic loss of CDKN2A were associated with favorable response. Even in Ph-negative ALL cell lines, IKZF1 deletion and bilallelic loss of CDKN2A were independently associated with higher BTZ sensitivity. BTZ showed only marginal cross-resistance to four representative chemotherapeutic agents (vincristine, dexamethasone, l-asparaginase, and daunorubicin) in B-cell precursor-ALL cell lines. To improve the efficacy and safety of proteasome inhibitor combination chemotherapy, we also analyzed the anti-leukemic activity of carfilzomib (CFZ), a second-generation proteasome inhibitor, as a substitute for BTZ. CFZ showed significantly higher activity than BTZ in the majority of ALL cell lines except for the P-glycoprotein-positive t(17;19) ALL cell lines, and IKZF1 deletion was also associated with a favorable response to CFZ treatment. P-glycoprotein inhibitors effectively restored the sensitivity to CFZ, but not BTZ, in P-glycoprotein-positive t(17;19) ALL cell lines. P-glycoprotein overexpressing ALL cell line showed a CFZ-specific resistance, while knockout of P-glycoprotein by genome editing with a CRISPR/Cas9 system sensitized P-glycoprotein-positive t(17;19) ALL cell line to CFZ. These observations suggested that IKZF1 deletion could be a useful biomarker to predict good sensitivity to CFZ and BTZ, and that CFZ combination chemotherapy may be a new therapeutic option with higher anti-leukemic activity for refractory ALL that contain P-glycoprotein-negative leukemia cells.

Lack of association between deletion polymorphism of BIM gene and in vitro drug sensitivity in B-cell precursor acute lymphoblastic leukemia

A deletion polymorphism in the BIM gene was identified as an intrinsic mechanism for resistance to tyrosine kinase inhibitor in chronic myeloid leukemia patients in East Asia. BIM is also involved in the responses to glucocorticoid and chemotherapy in acute lymphoblastic leukemia (ALL), suggesting a possible association between deletion polymorphism of BIM and the chemosensitivity of ALL. Thus, we analyzed 72 B-cell precursor (BCP)-ALL cell lines established from Japanese patients. Indeed, higher BIM gene expression was associated with good in vitro sensitivities to glucocorticoid and chemotherapeutic agents used in induction therapy. We also analyzed the methylation status of the BIM gene promoter by next generation sequencing of genome bisulfite PCR products, since genetic polymorphism could be insignificant when epigenetically inactivated. Hypermethylation of the BIM gene promoter was associated with lower BIM gene expression and poorer sensitivity to vincristine. Of note, however, the prevalence of a deletion polymorphism was not associated with the BIM gene expression level or drug sensitivities in BCP-ALL cell lines, in which the BIM gene was unmethylated. These observations suggest that an association of a deletion polymorphism of BIM and the response to induction therapy in BCP-ALL may be clinically minimal.

T315I mutation of BCR-ABL1 into human Philadelphia chromosome-positive leukemia cell lines by homologous recombination using the CRISPR/Cas9 system

In many cancers, somatic mutations confer tumorigenesis and drug-resistance. The recently established clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a potentially elegant approach to functionally evaluate mutations in cancers. To reproduce mutations by homologous recombination (HR), the HR pathway must be functional, but DNA damage repair is frequently impaired in cancers. Imatinib is a tyrosine kinase inhibitor for BCR-ABL1 in Philadelphia chromosome-positive (Ph+) leukemia, and development of resistance due to kinase domain mutation is an important issue. We attempted to introduce the T315I gatekeeper mutation into three Ph+ myeloid leukemia cell lines with a seemingly functional HR pathway due to resistance to the inhibitor for poly (ADP) ribose polymerase1. Imatinib-resistant sublines were efficiently developed by the CRISPR/Cas9 system after short-term selection with imatinib; resulting sublines acquired the T315I mutation after HR. Thus, the usefulness of CRISPR/Cas9 system for functional analysis of somatic mutations in cancers was demonstrated.

Splicing variant profiles and single nucleotide polymorphisms of the glucocorticoid receptor gene in relation to glucocorticoid sensitivity of B‐cell precursor acute lymphoblastic leukaemia

Glucocorticoid (GC) shows antileukaemic activity via binding to the GC receptor (GR). The human GR gene has 4 splicing variants besides the functional isoform GRα, but their significance in GC sensitivity of acute lymphoblastic leukaemia (ALL) has been inconsistent. Additionally, several studies evaluated the relevance of GR gene single nucleotide polymorphisms (SNPs) in the GC sensitivity of ALL, but the current cumulative evidence appears inconclusive. Addressing limitations in previous studies, we used a large series of B‐cell precursor ALL (BCP‐ALL) cell lines established from Japanese patients to comprehensively examine all 5 splicing variants of the GR gene and candidate SNPs, and their association with GC‐sensitivity. We performed real‐time reverse transcription polymerase chain reaction (RT‐PCR) analyses with 10 sets of primers that differentially quantify the 5 isoforms in different combinations, and the strongest correlations with GC sensitivity were observed for the real‐time RT‐PCR of exons 7 and 8 (prednisolone sensitivity; r = −0.534, R2 = 0.29, P = 1.4 × 10−6) and exons 8 and 9a (r = −0.583, R2 = 0.34, P = 7.6 × 10−8), both specific for GRα and GRγ isoforms. In contrast, the real‐time RT‐PCR of junction of exons 3g and 4 and exon 4, specific for GRγ isoform alone, did not show significant correlation with GC sensitivity (prednisolone sensitivity; r = −0.403, R2 = 0.16, P = 4.6 × 10−4). These observations are consistent with the notion that GRα plays a central role in the GC‐mediated proapoptotic activity in BCP‐ALL. In addition, a promoter region SNP genotype (rs72555796) showed a significant association with GC sensitivity (prednisolone sensitivity; P = .010) and tended to show an association with GR gene expression (RT‐PCR of exons 7 and 8; P = .170). These findings indicate that isoform profiles and SNP genotypes of the GR gene may be useful indicators of GC sensitivity in BCP‐ALL.

Erythrophagocytosis in T-cell type acute lymphoblastic leukaemia with near-tetraploidy

Erythrophagocytosis by leukaemic blasts—a poor prognostic feature—is mostly seen in acute myeloid leukaemia (AML), particularly monocytic leukaemia with cytogenetic abnormalities involving t(8;16)(p11;p13); MOZ-CBP or t(16;21)(p11;q22); FUS/TLS-ERG .1 This feature was reported in poorly differentiated leukaemias, including AML-M0, undifferentiated-type leukaemia and mixed lineage-type leukaemia, but very rarely in lineage-determined leukaemia, especially T-cell acute lymphoblastic leukaemia (T-ALL). We present a case of T-ALL with leukaemic blasts showing erythrophagocytosis, which was successfully treated with unrelated cord blood transplantation (uCBT). A 11-year-old female child presented with prolonged abdominal distention of 1 month duration. Physical examination revealed both liver and spleen edges at 15 cm below the right and left costal margins, respectively. Abdominal X-ray showed a large mass with displaced intestines and MRI confirmed significant hepatosplenomegaly on low-intensity and high-intensity T1-enhanced and T2-enhanced images, respectively. There was no thymus enlargement. Laboratory examinations of peripheral blood showed the following: white blood cell count, 1.92×109/L with no leukaemic blasts; haemoglobin, 11.4 g/dL; platelet count, 80×109/L and serum lactate dehydrogenase, 541 IU/L. There was no serological evidence of Epstein-Barr virus or cytomegalovirus infection. Morphological evaluation of a bone marrow smear using May-Giemsa staining showed approximately 70% leukaemic blasts with irregular nuclear membranes, few nucleoli and scanty cytoplasm with some vacuoles, classified as L2 according to the French-American-British (FAB) classification; 4.8% of leukaemic blasts showed erythrophagocytosis (figure 1A–D). All blasts were negative for myeloperoxidase and double esterase staining. Immunophenotypic analysis of the surface markers revealed the leukaemic blasts to be positive for CD2, CD3, CD4, CD5, CD7, TCR α/β, cyCD3, CD117, CD58 and CD99 …

Effective eculizumab therapy followed by BMT in a boy with paroxysmal nocturnal hemoglobinuria

A 9‐year‐old boy with paroxysmal nocturnal hemoglobinuria/aplastic anemia syndrome (PNH/AA) developed hemolytic crisis after receiving immunosuppressive therapy. Eculizumab dramatically relieved the signs and symptoms and then he safely underwent unrelated bone marrow transplantation, suggesting the feasibility and effectiveness of eculizumab before stem cell transplantation in children with PNH/AA in hemolytic crisis.

Pre-B-cell acute lymphoblastic leukemia in a boy with hereditary multiple exostoses caused by EXT1 deletion.

Patients with hereditary multiple exostoses have increased risk for malignant transformation of osteochondromas to secondary chondrosarcomas[1]. Previously, the association between hereditary multiple exostoses and acute myeloblastic leukemia was implicated in an 8-year-old girl in the journal [2]; however, molecular genetic analysis was not performed in that patient. We experienced the second case associated with leukemia and hereditary multiple exostoses. A male patient was born to nonconsanguineous Japanese parents (a 48-year-old father and a 39-year-old mother) at 40 weeks of gestation. He is the second child of these individuals. The mother had two sons with her ex-husband. All of his family members were healthy. At the age of 14 months, the patient was admitted to our hospital because of pre-B-cell precursor ALL. Among 19 bone marrow cells, one showed a 46,XY,t(1;19)(q23;p13) karyotype, whereas the remaining cells were normal. An RT-PCR assay detected chimeric E2a/Pbx1 transcripts. He was treated with the high-risk regimen of the Tokyo Children’s Cancer Study Group L99–1502 protocol, and he achieved complete remission at the age of 2 years. At the age of 6 years, protrusion of the right knee, pectus excavatum, and mild scoliosis were noted. A skeletal survey detected deformities of a right ulnar end and bilateral femoral epiphysis. No cone-shaped epiphysis was observed. He was diagnosed with multiple exostoses. GTG-banded chromosome analysis of peripheral lymphocytes revealed a 46,XY,del(8)(q24.1q24.21) karyotype. We asked the parents of the patient for their karyotype analysis, but they refused it. Array comparative genomic hybridization (CGH) was performed using the Illumina Omni1-Quad CNVTM platform (Illumina, Inc., San Diego, CA) with high-density oligonucleotides and content sourced from the UCSC hg18 human genome (NCBI build 36, March 2006) according to the Illumina “infinium assay” protocol (Illumina Inc.). Array CGH showed the presence of a 9.95-Mb deletion between 8q24.11 and 8q24.21 (chr8: 117,914,100–127,859,109) that encompassed EXT1 (located at chr8: 118,806,729–119,124,092), as well as a 0.61-MB duplication at