Mio Yano

Characterization of pediatric Philadelphia-negative B-cell precursor acute lymphoblastic leukemia with kinase fusions in Japan

Recent studies revealed that a substantial proportion of patients with high-risk B-cell precursor acute lymphoblastic leukemia (BCP-ALL) harbor fusions involving tyrosine kinase and cytokine receptors, such as ABL1, PDGFRB, JAK2 and CRLF2, which are targeted by tyrosine kinase inhibitors (TKIs). In the present study, transcriptome analysis or multiplex reverse transcriptase–PCR analysis of 373 BCP-ALL patients without recurrent genetic abnormalities identified 29 patients with kinase fusions. Clinically, male predominance (male/female: 22/7), older age at onset (mean age at onset: 8.8 years) and a high white blood cell count at diagnosis (mean: 94 200/μl) reflected the predominance of National Cancer Institute high-risk (NCI-HR) patients (NCI-standard risk/HR: 8/21). Genetic analysis identified three patients with ABL1 rearrangements, eight with PDGFRB rearrangements, two with JAK2 rearrangements, three with IgH-EPOR and one with NCOR1-LYN. Of the 14 patients with CRLF2 rearrangements, two harbored IgH-EPOR and PDGFRB rearrangements. IKZF1 deletion was present in 16 of the 22 patients. The 5-year event-free and overall survival rates were 48.6±9.7% and 73.5±8.6%, respectively. The outcome was not satisfactory without sophisticated minimal residual disease-based stratification. Furthermore, the efficacy of TKIs combined with conventional chemotherapy without allogeneic hematopoietic stem cell transplantation in this cohort should be determined.

Sensitivity of MLL -rearranged AML cells to all- trans retinoic acid is associated with the level of H3K4me2 in the RARα promoter region

All-trans retinoic acid (ATRA) is well established as differentiation therapy for acute promyelocytic leukemia (APL) in which the PML–RARα (promyelocytic leukemia-retinoic acid receptor α) fusion protein causes blockade of the retinoic acid (RA) pathway; however, in types of acute myeloid leukemia (AML) other than APL, the mechanism of RA pathway inactivation is not fully understood. This study revealed the potential mechanism of high ATRA sensitivity of mixed-lineage leukemia (MLL)-AF9-positive AML compared with MLL-AF4/5q31-positive AML. Treatment with ATRA induced significant myeloid differentiation accompanied by upregulation of RARα, C/EBPα, C/EBPɛ and PU.1 in MLL-AF9-positive but not in MLL-AF4/5q31-positive cells. Combining ATRA with cytarabine had a synergistic antileukemic effect in MLL-AF9-positive cells in vitro. The level of dimethyl histone H3 lysine 4 (H3K4me2) in the RARα gene-promoter region, PU.1 upstream regulatory region (URE) and RUNX1+24/+25 intronic enhancer was higher in MLL-AF9-positive cells than in MLL-AF4-positive cells, and inhibiting lysine-specific demethylase 1, which acts as a histone demethylase inhibitor, reactivated ATRA sensitivity in MLL-AF4-positive cells. These findings suggest that the level of H3K4me2 in the RARα gene-promoter region, PU.1 URE and RUNX1 intronic enhancer is determined by the MLL-fusion partner. Our findings provide insight into the mechanisms of ATRA sensitivity in AML and novel treatment strategies for ATRA-resistant AML.

An overall characterization of pediatric acute lymphoblastic leukemia with CRLF2 overexpression

For an overall characterization of pediatric B‐cell precursor acute lymphoblastic leukemia (BCPALL) with CRLF2 overexpression (OE), we conducted genetic analysis of CRLF2 in 167 pediatric BCPALL patients. CRLF2 OE was detected in 30 (18%) of 167 patients, the P2RY8‐CRLF2 fusion was identified in only 3 (1.8%) of 167 patients, all of which demonstrated CRLF2 OE. Moreover, CRLF2 gain was identified in 18 (11%) of 167 patients. Messenger RNA sequencing revealed a novel fusion transcript, CSF2RA‐CRLF2, in a case with CRLF2 OE, suggesting that this fusion is associated with CRLF2 OE. In survival analysis, no significant differences in 5‐year event‐free survival (EFS) and overall survival were observed between patients with and without CRLF2 OE (70.7 vs. 75.4%, log rank P = 0.68 and 96.4 vs. 82.1%, log rank P = 0.11, respectively). However, a significant difference in 5‐year EFS between CRLF2 OE patients with and without IKZF1 deletion was observed (44.4 vs. 83.1%, log rank P = 0.02). In multivariate analysis, only IKZF1 deletion was a significant predictor of inferior OS (hazard ratio: 2.427, P = 0.04).These findings suggest that CRLF2 OE is not an independent prognostic factor in pediatric BCPALL.

Identification of novel kinase fusion transcripts in paediatric B cell precursor acute lymphoblastic leukaemia with IKZF1 deletion

Activating tyrosine kinase mutations or cytokine receptor signalling alterations have attracted attention as therapeutic targets for high‐risk paediatric acute lymphoblastic leukaemia (ALL). We identified two novel kinase fusions, OFD1‐JAK2 and NCOR1‐LYN, in paediatric ALL patients with IKZF1 deletion, by mRNA sequencing. The patient with CSF2RA‐CRLF2 also harboured IGH‐EPOR. All these patients had high‐risk features, such as high initial white blood cell counts and initial poor response to prednisolone. The functional analysis of these novel fusions is on‐going to determine whether these genetic alterations can be targeted by drugs.

BCL2 Inhibitor (ABT-737): A Restorer of Prednisolone Sensitivity in Early T-Cell Precursor-Acute Lymphoblastic Leukemia with High MEF2C Expression?

Early T-cell precursor-acute lymphoblastic leukemia (ETP-ALL) has been identified as a high-risk subtype of pediatric T-cell acute lymphoblastic leukemia (T-ALL). Conventional chemotherapy is not fully effective for this subtype of leukemia; therefore, potential therapeutic targets need to be explored. Analysis of the gene expression patterns of the transcription factors in pediatric T-ALL revealed that MEF2C and FLT3 were expressed at higher levels in ETP-ALL than typical T-ALL. Using human T-ALL and BaF3 cell lines with high expression levels of MEF2C, the present study tested whether the BCL2 inhibitor (ABT-737) restores the sensitivity to prednisolone (PSL), because MEF2C causes PSL resistance, possibly by augmenting the anti-apoptotic activity of BCL2. Treatment with PSL and ABT-737 caused a significant reduction in the IC50 of PSL in the MEF2C-expressing LOUCY cells, in addition to the MEF2C-transduced BaF3 cells, but not in the non-MEF2C-expressing Jurkat cells. The combination treatment significantly accelerated the killing of primary leukemic blast cells of ETP-ALL with high expression levels of MEF2C, which were co-cultured with murine stromal cells. These findings suggest that BCL2 inhibitors may be a therapeutic candidate in vivo for patients with ETP-ALL with high expression levels of MEF2C.

Ph-like acute lymphoblastic leukemia with a novel PAX5-KIDINS220 fusion transcript.

Although “paired box 5” (PAX5)‐related fusion genes are well documented in childhood B‐cell precursor acute lymphoblastic leukemia (ALL), these types of fusion with the exception of PAX5‐JAK2 are rarely seen in patients with gene expression profiles similar to those of BCR‐ABL1 (Philadelphia)‐positive ALL (Ph‐like ALL). We report a novel fusion of the genes PAX5 and “kinase D‐interacting substrate of 220 kDa” (KIDINS220, also known as ARMS) in a Ph‐like ALL. As PAX5 is a master regulator of B‐lymphocyte differentiation, PAX5 rearrangements induce a differentiation block in B lymphocytes. KIDINS220 is a mediator of multiple receptor signaling pathways, interacts with both T‐ and B‐cell receptors, and is necessary for sustained extracellular signal‐regulated kinase (ERK) signaling. Although functional studies are needed, the PAX5‐KIDINS220 fusion protein might not only inhibit wild‐type PAX5 function, but also promote sustained activation of the ERK signaling pathway through upregulation of KIDINS220.

IKZF1 deletion is enriched in pediatric B-cell precursor acute lymphoblastic leukemia patients showing prednisolone resistance

IKZF1 deletion is enriched in pediatric B-cell precursor acute lymphoblastic leukemia patients showing prednisolone resistance

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.

Clinical significance of SH2B3 (LNK) expression in paediatric B‐cell precursor acute lymphoblastic leukaemia

Aberrant JAK-STAT signalling is a key mechanism involved in the development of paediatric B-cell precursor acute lymphoblastic leukaemia (B-ALL). Gene alterations that lead to aberrant JAK-STAT signalling, such as in JAK2, CRLF2 and IL7R, have been reported (Mullighan et al, 2009). However, the JAK2 activating mutation is extremely rare and the prognostic significance of CRLF2 alterations for Japanese cohorts is controversial (Asai et al, 2013). It is therefore likely that there are other factors associated with the abnormal activation of JAK-STAT signalling. The adaptor protein SH2B3 (also termed LNK) is a negative regulator of multiple cytokine signalling molecules and tyrosine kinase receptors with important roles in the homeostasis of haematopoietic stem cells and lymphoid progenitors (Gueller et al, 2008; Baran-Marszak et al, 2010; Lin et al, 2012). Previous studies have revealed that Sh2b3 (Lnk)-deficient mice have marked expansion of B cells in response to stem cell factor stimulation, indicating that SH2B3 has an important role in the control of B-cell homeostasis (Takaki et al, 2003). Additionally, in paediatric ALL, recent genomewide analyses and next-generation sequencing approaches have identified not only somatic mutations of SH2B3 with low frequency in high-risk B-ALL (Roberts et al, 2012) but also germline SH2B3 mutation in a patient with B-ALL (Perez-Garcia et al, 2013). These findings suggest that loss of function of SH2B3 might be highly associated with leukaemogenesis in B-ALL. Therefore, we analysed cases of paediatric B-ALL to determine the prognostic impact of SH2B3 gene expression. The study samples comprised mononuclear cells isolated from diagnostic bone marrow or peripheral blood from 164 paediatric B-ALL patients treated with the Japan Association of Childhood Leukemia Study (JACLS) ALL-02 protocol (Yoshida et al, 2015), and from peripheral blood from 20 healthy volunteers. Informed consent was obtained from the patients’ guardians, according to the Declaration of Helsinki. Treatment and genetic study protocols were approved by the institutional review boards of the participating institutes. SH2B3 mRNA expression level was determined by realtime quantitative-polymerase chain reaction (q-PCR), using the comparative threshold (ΔCT) method. The primer pairs used in this study are listed in Table SI. The cut-off value of the SH2B3 expression level was determined using receiver operating characteristic (ROC) analysis. Based on this cut-off value, estimation of survival distributions was performed by the Kaplan–Meier method and differences compared using a log-rank test. Hazard ratios for probability of relapse between subgroups were calculated using a univariate Cox model. Multivariate analysis was performed using a Cox regression model. The characteristics of the 164 patients in the study are summarized in Table SII. Sixty-two patients did not respond to the initial prednisolone therapy and were treated according to the ALL-02 extremely high-risk (ER) protocol. The remaining 102 patients showed good response to initial prednisolone and were treated according to the ALL-02 high-risk (HR) protocol. The frequency of IKZF1 deletion was simlar in the two risk groups. In contrast, three patients with P2RY8-CRLF2 were present only in ER group (Table SII). None of the patients with IKZF1 deletion (n = 25) harboured JAK2 activating mutation. q-PCR analysis determined that SH2B3 mRNA expression levels in the mononuclear cells from paediatric B-ALL patient samples were significantly lower than in those from healthy volunteers (P < 0 01; Figure S1A). Among the 164 B-ALL cases, SH2B3 expression was lower in relapsed or induction failure (IF) patients (n = 36) than that in the remaining patients who maintained complete remission, but the difference was not statistically significant (P = 0 067). On the other hand, SH2B3 expression was significantly lower in dead patients (P < 0 01; Figure S1B, C). When the 164 patients were split into two risk groups based on initial prednisolone response and treatment protocol, the SH2B3 expression was significantly lower in the relapsed or IF patients, in both the HR (n = 102) and ER (n = 62) groups (P < 0 05 for both; Fig 1A, B). These findings suggested that the expression level of SH2B3 is a prognostic marker in B-ALL. In both risk groups, 5-year event-free survival (EFS) was significantly better in patients with high SH2B3 expression. In the HR group, 5-year EFS was 93 7% for those with high SH2B3 expression (95% confidence interval [CI], 87 6–99 7) versus 75 2% for those with low expression (95% CI, 60 9– 89 4; log rank P = 0 0142). In the ER group, 5-year EFS was 90 6% in those with high SH2B3 (95% CI, 80 4–100 vs. 50 1% for those with low SH2B3 expression (95% CI, 30 1– 70 1; log rank P = 0 0012; Figure S2A). Similarly, high SH2B3 expression contributed to a superior 5-year overall survival (OS) (HR group: 89 4%; 95% CI, 81 3–97 6 vs. 73 6%; 95% CI, 59 6–87 6; log rank P = 0 0256. ER group: 79 1%; 95% CI, 64 2–94 1 vs. 52 0%; 95% CI, 33 8–70 2; log Correspondence

Copy number abnormality of acute lymphoblastic leukemia cell lines based on their genetic subtypes

In this study, we performed genetic analysis of 83 B cell precursor acute lymphoblastic leukemia (B-ALL) cell lines. First, we performed multiplex ligation-dependent probe amplification analysis to identify copy number abnormalities (CNAs) in eight genes associated with B-ALL according to genetic subtype. In Ph+ B-ALL cell lines, the frequencies of IKZF1, CDKN2A/2B, BTG1, and PAX5 deletion were significantly higher than those in Ph− B-ALL cell lines. The frequency of CDKN2A/2B deletion in KMT2A rearranged cell lines was significantly lower than that in non-KMT2A rearranged cell lines. These findings suggest that CNAs are correlated with genetic subtype in B-ALL cell lines. In addition, we determined that three B-other ALL cell lines had IKZF1 deletions (YCUB-5, KOPN49, and KOPN75); we therefore performed comprehensive genetic analysis of these cell lines. YCUB-5, KOPN49, and KOPN75 had P2RY8-CRLF2, IgH-CRLF2, and PAX5-ETV6 fusions, respectively. Moreover, targeted capture sequencing revealed that YCUB-5 had JAK2 R683I and KRAS G12D, and KOPN49 had JAK2 R683G and KRAS G13D mutations. These data may contribute to progress in the field of leukemia research.