Tyrosine kinase inhibitor resistance in de novo BCR::ABL1-positive BCP-ALL beyond kinase domain mutations.

ABSTRACT: A better understanding of ABL1 kinase domain mutation-independent causes of tyrosine kinase inhibitor (TKI) resistance is needed for BCR::ABL1-positive B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Although TKIs have dramatically improved outcomes, a subset of patients still experiences relapsed or refractory disease. We aimed to identify potential biomarkers of intrinsic TKI resistance at diagnosis in samples from 32 pediatric and 19 adult patients with BCR::ABL1-positive BCP-ALL. Reduced ex vivo imatinib sensitivity was observed in cells derived from newly diagnosed patients who relapsed after combined TKI and chemotherapy treatment compared with cells derived from patients who remained in continuous complete remission. We observed that ex vivo imatinib resistance was inversely correlated with the amount of (phosphorylated) BCR::ABL1/ABL1 protein present in samples that were taken at diagnosis without prior TKI exposure. This suggests an intrinsic cause of TKI resistance that is independent of functional BCR::ABL1 signaling. Simultaneous deletions of IKZF1 and CDKN2A/B and/or PAX5 (IKZF1plus), as well as deletions of PAX5 alone, were related to ex vivo imatinib resistance. In addition, somatic lesions involving ZEB2, SETD2, SH2B3, and CRLF2 were associated with reduced ex vivo imatinib sensitivity. Our data suggest that the poor prognostic value of IKZF1(plus) deletions is linked to intrinsic mechanisms of TKI resistance other than ABL1 kinase domain mutations in newly diagnosed pediatric and adult BCR::ABL1-positive BCP-ALL.

Tyrosine kinase inhibitor response of ABL-class acute lymphoblastic leukemia: the role of kinase type and SH3 domain.

ABSTRACT: Acute lymphoblastic leukemia (ALL) with fusions of ABL-class tyrosine kinase genes other than BCR::ABL1 occurs in ∼3% of children with ALL. The tyrosine kinase genes involved in this BCR::ABL1-like (Ph-like) subtype include ABL1, PDGFRB, ABL2, and CSF1R, each of which has up to 10 described partner genes. ABL-class ALL resembles BCR::ABL1-positive ALL with a similar gene expression profile, poor response to chemotherapy, and sensitivity to tyrosine kinase inhibitors (TKIs). There is a lack of comprehensive data regarding TKI sensitivity in the heterogeneous group of ABL-class ALL. We observed variability in TKI sensitivity within and among each ABL-class tyrosine kinase gene subgroup. We showed that ALL samples with fusions for any of the 4 tyrosine kinase genes were relatively sensitive to imatinib. In contrast, the PDGFRB-fused ALL samples were less sensitive to dasatinib and bosutinib. Variation in ex vivo TKI response within the subset of samples with the same ABL-class tyrosine kinase gene was not associated with the ALL immunophenotype, 5' fusion partner, presence or absence of Src-homology-2/3 domains, or deletions of IKZF1, PAX5, or CDKN2A/B. In conclusion, the tyrosine kinase gene involved in ABL-class ALL is the main determinant of TKI sensitivity and relevant for specific TKI selection.

Integrating copy number data of 64 iAMP21 BCP-ALL patients narrows the common region of amplification to 1.57 Mb.

Background and purpose: Intrachromosomal amplification of chromosome 21 (iAMP21) is a rare subtype of B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). It is unknown how iAMP21 contributes to leukaemia. The currently known commonly amplified region is 5.1 Mb. Methods: We aimed to narrow down the common region of amplification by using high resolution techniques. Array comparative genomic hybridization (aCGH) was used to determine copy number aberrations, Affymetrix U133 Plus2 expression arrays were used to determine gene expression. Genome-wide expression correlations were evaluated using Globaltest. Results: We narrowed down the common region of amplification by combining copy number data from 12 iAMP21 cases with 52 cases from literature. The combined common region of amplification was 1.57 Mb, located from 36.07 to 37.64 Mb (GRCh38). This region is located telomeric from, but not including, RUNX1, which is the locus commonly used to diagnose iAMP21. This narrow region, which falls inside the Down Syndrome critical region, includes 13 genes of which the expression of eight genes was significantly upregulated compared with 143 non-iAMP21 B-other cases. Among these, transcriptional repressor RIPPLY3 (also known as DSCR6) was the highest overexpressed gene (fold change = 4.2, FDR < 0.001) and most strongly correlated (R = 0.58) with iAMP21-related genome-wide expression changes. Discussion: The more precise definition of the common region of amplification could be beneficial in the diagnosis of iAMP21 based on copy number analysis from DNA sequencing or arrays as well as stimulate functional research into the role of the included genes in iAMP21 biology.

Ibrutinib is not an effective drug in primografts of TCF3-PBX1.

AIM: The Bruton's tyrosine kinase (BTK) inhibitor Ibrutinib (PCI-32765) is effective in patients with multiple myeloma, non-Hodgkin lymphoma and chronic lymphoblastic leukemia. We previously showed that primary cells of children with TCF3-PBX1 positive B-cell precursor acute lymphoblastic leukemia (BCP-ALL) express BTK and are sensitive to ibrutinib in vitro. However, preclinical studies in mice are lacking that justify clinical implementation. METHODS: Immunocompromised NSG mice were engrafted with a luciferase-positive TCF3-PBX1 leukemic cell line or primary leukemic cells and treated with ibrutinib or placebo. Additionally, primary cells were exposed in vitro to 4 main induction drugs as monotherapy and in combination with ibrutinib. RESULTS: Treatment with ibrutinib of mice engrafted with a TCF3-PBX1 cell line, TCF3-PBX1 positive or TCF3-PBX1 negative primary leukemic cells did not result in prolonged life span compared to placebo treated mice. In vitro sensitivity to ibrutinib was unaltered in leukemic cells obtained from engrafted mice compared to the original material. However, ibrutinib treatment did not affect leukemic cell viability and tumor outgrowth, nor could lymphocytosis be detected. Ibrutinib was biologically active, since hCD19+ cells harvested from ibrutinib treated mice had no detectable levels of phospho-BTK at tyrosine 223 (pBTK Y223), whereas pBTK Y223 was still detectable in placebo treated cases. In combination tests, we noticed an antagonistic effect of ibrutinib on vincristine sensitivity, which was not observed for prednisolone, L-asparaginase and daunorubicin. CONCLUSIONS: We conclude that ibrutinib is not the precision medicine of choice for TCF3-PBX1 positive BCP-ALL.

Copy number alterations in B-cell development genes, drug resistance, and clinical outcome in pediatric B-cell precursor acute lymphoblastic leukemia.

Pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is associated with a high frequency of copy number alterations (CNAs) in IKZF1, EBF1, PAX5, CDKN2A/B, RB1, BTG1, ETV6, and/or the PAR1 region (henceforth: B-cell development genes). We aimed to gain insight in the association between CNAs in these genes, clinical outcome parameters, and cellular drug resistance. 71% of newly diagnosed pediatric BCP-ALL cases harbored one or more CNAs in these B-cell development genes. The distribution and clinical relevance of these CNAs was highly subtype-dependent. In the DCOG-ALL10 cohort, only loss of IKZF1 associated as single marker with unfavorable outcome parameters and cellular drug resistance. Prednisolone resistance was observed in IKZF1-deleted primary high hyperdiploid cells (~1500-fold), while thiopurine resistance was detected in IKZF1-deleted primary BCR-ABL1-like and non-BCR-ABL1-like B-other cells (~2.7-fold). The previously described risk stratification classifiers, i.e. IKZF1plus and integrated cytogenetic and CNA classification, both predicted unfavorable outcome in the DCOG-ALL10 cohort, and associated with ex vivo drug cellular resistance to thiopurines, or L-asparaginase and thiopurines, respectively. This resistance could be attributed to overrepresentation of BCR-ABL1-like cases in these risk groups. Taken together, our data indicate that the prognostic value of CNAs in B-cell development genes is linked to subtype-related drug responses.

JAK2 aberrations in childhood B-cell precursor acute lymphoblastic leukemia.

JAK2 abnormalities may serve as target for precision medicines in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In the current study we performed a screening for JAK2 mutations and translocations, analyzed the clinical outcome and studied the efficacy of two JAK inhibitors in primary BCP-ALL cells. Importantly, we identify a number of limitations of JAK inhibitor therapy. JAK2 mutations mainly occurred in the poor prognostic subtypes BCR-ABL1-like and non- BCR-ABL1-like B-other (negative for sentinel cytogenetic lesions). JAK2 translocations were restricted to BCR-ABL1-like cases. Momelotinib and ruxolitinib were cytotoxic in both JAK2 translocated and JAK2 mutated cells, although efficacy in JAK2 mutated cells highly depended on cytokine receptor activation by TSLP. However, our data also suggest that the effect of JAK inhibition may be compromised by mutations in alternative survival pathways and microenvironment-induced resistance. Furthermore, inhibitors induced accumulation of phosphorylated JAK2Y1007, which resulted in a profound re-activation of JAK2 signaling upon release of the inhibitors. This preclinical evidence implies that further optimization and evaluation of JAK inhibitor treatment is necessary prior to its clinical integration in pediatric BCP-ALL.

Tyrosine kinase fusion genes in pediatric BCR-ABL1-like acute lymphoblastic leukemia.

Approximately 15% of pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL) is characterized by gene expression similar to that of BCR-ABL1-positive disease and unfavorable prognosis. This BCR-ABL1-like subtype shows a high frequency of B-cell development gene aberrations and tyrosine kinase-activating lesions. To evaluate the clinical significance of tyrosine kinase gene fusions in children with BCP-ALL, we studied the frequency of recently identified tyrosine kinase fusions, associated genetic features, and prognosis in a representative Dutch/German cohort. We identified 14 tyrosine kinase fusions among 77 BCR-ABL1-like cases (18%) and none among 76 non-BCR-ABL1-like B-other cases. Novel exon fusions were identified for RCSD1-ABL2 and TERF2-JAK2. JAK2 mutation was mutually exclusive with tyrosine kinase fusions and only occurred in cases with high CRLF2 expression. The non/late response rate and levels of minimal residual disease in the fusion-positive BCR-ABL1-like group were higher than in the non-BCR-ABL1-like B-others (p<0.01), and also higher, albeit not statistically significant, compared with the fusion-negative BCR-ABL1-like group. The 8-year cumulative incidence of relapse in the fusion-positive BCR-ABL1-like group (35%) was comparable with that in the fusion-negative BCR-ABL1-like group (35%), and worse than in the non-BCR-ABL1-like B-other group (17%, p=0.07). IKZF1 deletions, predominantly other than the dominant-negative isoform and full deletion, co-occurred with tyrosine kinase fusions. This study shows that tyrosine kinase fusion-positive cases are a high-risk subtype of BCP-ALL, which warrants further studies with specific kinase inhibitors to improve outcome.