Chiara Palmi

The TEL-AML1 leukemia fusion gene dysregulates the TGF-β pathway in early B lineage progenitor cells

Chromosome translocation to generate the TEL-AML1 (also known as ETV6-RUNX1) chimeric fusion gene is a frequent and early or initiating event in childhood acute lymphoblastic leukemia (ALL). Our starting hypothesis was that the TEL-AML1 protein generates and maintains preleukemic clones and that conversion to overt disease requires secondary genetic changes, possibly in the context of abnormal immune responses. Here, we show that a murine B cell progenitor cell line expressing inducible TEL-AML1 proliferates at a slower rate than parent cells but is more resistant to further inhibition of proliferation by TGF-beta. This facilitates the competitive expansion of TEL-AML1-expressing cells in the presence of TGF-beta. Further analysis indicated that TEL-AML1 binds to a principal TGF-beta signaling target, Smad3, and compromises its ability to activate target promoters. In mice expressing a TEL-AML1 transgene, early, pre-pro-B cells were increased in number and also showed reduced sensitivity to TGF-beta-mediated inhibition of proliferation. Moreover, expression of TEL-AML1 in human cord blood progenitor cells led to the expansion of a candidate preleukemic stem cell population that had an early B lineage phenotype (CD34+CD38-CD19+) and a marked growth advantage in the presence of TGF-beta. Collectively, these data suggest a plausible mechanism by which dysregulated immune responses to infection might promote the malignant evolution of TEL-AML1-expressing preleukemic clones.

Poor prognosis for P2RY8-CRLF2 fusion but not for CRLF2 over-expression in children with intermediate risk B-cell precursor acute lymphoblastic leukemia

Pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has achieved an 80% cure rate as a result of a risk-adapted therapy largely based on minimal residual disease (MRD) monitoring. However, relapse is still the most frequent adverse event, occurring mainly in the patients with intermediate MRD levels (intermediate risk, IR), emphasizing the need for new prognostic markers. We analyzed the prognostic impact of cytokine receptor-like factor 2 (CRLF2) over-expression and P2RY8-CRLF2 fusion in 464 BCP-ALL patients (not affected by Down syndrome and BCR-ABL negative) enrolled in the AIEOP-BFM ALL2000 study in Italy. In 22/464 (4.7%) samples, RQ-PCR showed CRLF2 over-expression (⩾20 times higher than the overall median). P2RY8-CRLF2 fusion was detected in 22/365 (6%) cases, with 10/22 cases also showing CRLF2 over-expression. P2RY8-CRLF2 fusion was the most relevant prognostic factor independent of CRLF2 over-expression with a threefold increase in risk of relapse. Significantly, the cumulative incidence of relapse of the P2RY8-CRLF2+ patients in the IR group was high (61.1%±12.9 vs 17.6%±2.6, P<0.0001), similar to high-risk patients in AIEOP-BFM ALL2000 study. These results were confirmed in a cohort of patients treated in Germany. In conclusion, P2RY8-CRLF2 identifies a subset of BCP-ALL patients currently stratified as IR that could be considered for treatment intensification.

PAX5/TEL acts as a transcriptional repressor causing down-modulation of CD19, enhances migration to CXCL12, and confers survival advantage in pre-BI cells

PAX5 is a transcription factor essential for B-cell development. Recently, it has been found as a frequent target of aberrancies in childhood acute lymphoblastic leukemia (ALL; 30% of B cell ALL cases), showing monoallelic loss, point mutations, or chromosomal translocations. The role of these aberrancies is still poorly understood. We previously cloned the PAX5/TEL fusion gene in a patient affected by B-cell precursor ALL with a t(9;12) translocation. This is the first report investigating the molecular and functional roles of PAX5/TEL protein in vitro from murine wild-type pre-BI cells. We showed that PAX5/TEL protein acts as an aberrant transcription factor with repressor function, recruiting mSin3A, down-regulating B220, CD19, BLNK, MB-1, FLT3, and mu heavy chain expression, thus suggesting a block on B-cell differentiation. In a PAX5-deficient context, the presence of PAX5/TEL did not replace PAX5 functions. PAX5/TEL protein enhances cell migration towards CXCL12, with the overexpression of CXCR4. Moreover, the presence of the fusion gene overcomes interleukin-7 withdrawal and interferes with transforming growth factor-beta1 pathway, inducing resistance and conferring cells an advantage in proliferation and survival. Thus, in vitro, the PAX5/TEL protein has a dominant effect on wild-type PAX5, interferes with the process of B-cell differentiation and migration, and induces resistance to apoptosis. Taken together, these phenomena likely represent key events in the process of B-cell transformation.

What is the relevance of Ikaros gene deletions as a prognostic marker in pediatric Philadelphia-negative B-cell precursor acute lymphoblastic leukemia?

New prognostic markers are needed for upfront identification of patients with acute lymphocytic leukemia with a high risk of relapse or who are not likely to respond to the most aggressive chemotherapy. We focused our analysis on Ikaros (IKZF1) gene deletions in a homogeneous cohort of 410 pediatric patients with Philadelphia chromosome-negative, B-cell precursor acute lymphoblastic leukemia enrolled in Italy into the AIEOP-BFM ALL2000 study. We confirm their reported poor prognostic value, although the associated event-free survival was relatively high (approximately 70%). The difference in the cumulative incidence of relapse between patients positive or not for IKZF1 deletions was not marked: 24.2% (5.9) versus 13.1% (1.8) overall and 23.9% (6.6) versus 16.5% (2.5) in the intermediate-risk subgroup. In line with this, IKZF1 deletions were not an independent prognostic factor for the hazard of relapse. Most IKZF1-deleted cases stratified in the high-risk group relapsed, suggesting that once identified, patients with these deletions require an alternative treatment. In conclusion, the need of and benefit from introducing IKZF1 deletions as an additional stratification marker for patients with Philadelphia-negative B-cell precursor acute lymphoblastic leukemia remain questionable.

Treatment outcome of CRLF2-rearranged childhood acute lymphoblastic leukaemia: a comparative analysis of the AIEOP-BFM and UK NCRI-CCLG study groups.

The prognostic relevance of CRLF2 ‐rearrangements in childhood acute B‐cell precursor lymphoblastic leukaemia (ALL), was assessed by a comparative analysis of 114 non‐Down‐syndrome patients (99 P2RY8‐CRLF2+ , 15 IGH@‐CRLF2+ ), 76 from the AIEOP‐BFM ALL 2000 and 38 from the MRC ALL97 trials. The 6‐year cumulative relapse incidence of P2RY8‐CRLF2+ patients treated on the two trials was not statistically different: 0·37 ± 0·06 vs. 0·25 ± 0·08 (P = 0·194). In contrast, 0/9 IGH@‐CRLF2+ AIEOP‐BFM, but 5/6 ALL97 patients relapsed. Conclusively, P2RY8‐CRLF2+ patients had an intermediate protocol‐independent outcome while the different prognosis of IGH@‐CRLF2+ patients could be related to the different structures of the applied treatment protocols.

Prognostic value of rare IKZF1 deletion in childhood B-cell precursor acute lymphoblastic leukemia: An international collaborative study

Deletions in IKZF1 are found in ~15% of children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). There is strong evidence for the poor prognosis of IKZF1 deletions affecting exons 4–7 and exons 1–8, but evidence for the remaining 33% of cases harboring other variants of IKZF1 deletions is lacking. In an international multicenter study we analyzed the prognostic value of these rare variants in a case–control design. Each IKZF1-deleted case was matched to three IKZF1 wild-type controls based on cytogenetic subtype, treatment protocol, risk stratification arm, white blood cell count and age. Hazard ratios for the prognostic impact of rare IKZF1 deletions on event-free survival were calculated by matched pair Cox regression. Matched pair analysis for all 134 cases with rare IKZF1 deletions together revealed a poor prognosis (P<0.001) that was evident in each risk stratification arm. Rare variant types with the most unfavorable event-free survival were DEL 2–7 (P=0.03), DEL 2–8 (P=0.002) and DEL-Other (P<0.001). The prognosis of each type of rare variant was equal or worse compared with the well-known major DEL 4–7 and DEL 1–8 IKZF1 deletion variants. We therefore conclude that all variants of rare IKZF1 deletions are associated with an unfavorable prognosis in pediatric BCP-ALL.

PAX5/ETV6 alters the gene expression profile of precursor B cells with opposite dominant effect on endogenous PAX5.

PAX5/ETV6 alters the gene expression profile of precursor B cells with opposite dominant effect on endogenous PAX5

Impact of IKZF1 deletions on IKZF1 expression and outcome in Philadelphia chromosome negative childhood BCP-ALL. Reply to “Incidence and biological significance of IKZF1/Ikaros gene deletions in pediatric Philadelphia chromosome negative and Philadelphia chromosome positive B-cell precursor acute lymphoblastic leukemia”

We thank Dr. Qazi and Dr. Uckun for their letter that focused on the biological relevance of IKZF1 deletions in pediatric acute lymphoblastic leukemia (ALL), and in particular on the lack of correlation between IKZF1 deletions and the expected deregulation of IKZF1 expression. This information

Suppressors and activators of JAK-STAT signaling at diagnosis and relapse of acute lymphoblastic leukemia in Down syndrome

Significance Children with Down syndrome are at increased risk for B-cell acute lymphoblastic leukemia (DS-ALL), often expressing cytokine receptor-like factor 2 (CRLF2). Here we studied matched diagnosis and relapse DS-ALLs to understand the pathogenesis of relapse. We confirm that enhanced JAK-STAT signaling frequently “drives” CRLF2pos DS-ALL at diagnosis, but discovered that clones with JAK mutations are unstable, suggesting that they also endowed the transformed cells with vulnerabilities. We find USP9X loss in up to 25% of CRLF2pos ALLs, and demonstrate that its ablation decreases the toxic effect of JAK2 hypersignaling. Thus, in CRLF2pos ALLs JAK-STAT signaling is often buffered by loss of USP9X. These results have therapeutic implications because they suggest that ALL cells can tolerate a limited range of JAK-STAT signaling. Children with Down syndrome (DS) are prone to development of high-risk B-cell precursor ALL (DS-ALL), which differs genetically from most sporadic pediatric ALLs. Increased expression of cytokine receptor-like factor 2 (CRLF2), the receptor to thymic stromal lymphopoietin (TSLP), characterizes about half of DS-ALLs and also a subgroup of sporadic “Philadelphia-like” ALLs. To understand the pathogenesis of relapsed DS-ALL, we performed integrative genomic analysis of 25 matched diagnosis-remission and -relapse DS-ALLs. We found that the CRLF2 rearrangements are early events during DS-ALL evolution and generally stable between diagnoses and relapse. Secondary activating signaling events in the JAK-STAT/RAS pathway were ubiquitous but highly redundant between diagnosis and relapse, suggesting that signaling is essential but that no specific mutations are “relapse driving.” We further found that activated JAK2 may be naturally suppressed in 25% of CRLF2pos DS-ALLs by loss-of-function aberrations in USP9X, a deubiquitinase previously shown to stabilize the activated phosphorylated JAK2. Interrogation of large ALL genomic databases extended our findings up to 25% of CRLF2pos, Philadelphia-like ALLs. Pharmacological or genetic inhibition of USP9X, as well as treatment with low-dose ruxolitinib, enhanced the survival of pre-B ALL cells overexpressing mutated JAK2. Thus, somehow counterintuitive, we found that suppression of JAK-STAT “hypersignaling” may be beneficial to leukemic B-cell precursors. This finding and the reduction of JAK mutated clones at relapse suggest that the therapeutic effect of JAK specific inhibitors may be limited. Rather, combined signaling inhibitors or direct targeting of the TSLP receptor may be a useful therapeutic strategy for DS-ALL.

The histone deacetylase inhibitor givinostat (ITF2357) exhibits potent anti-tumor activity against CRLF2 -rearranged BCP-ALL

Leukemias bearing CRLF2 and JAK2 gene alterations are characterized by aberrant JAK/STAT signaling and poor prognosis. The HDAC inhibitor givinostat/ITF2357 has been shown to exert anti-neoplastic activity against both systemic juvenile idiopathic arthritis and myeloproliferative neoplasms through inhibition of the JAK/STAT pathway. These findings led us to hypothesize that givinostat might also act against CRLF2-rearranged BCP-ALL, which lack effective therapies. Here, we found that givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged cell lines, positive for exon 16 JAK2 mutations. Likewise, givinostat killed primary cells, but not their normal hematopoietic counterparts, from patients carrying CRLF2 rearrangements. At low doses, givinostat downregulated the expression of genes belonging to the JAK/STAT pathway and inhibited STAT5 phosphorylation. In vivo, givinostat significantly reduced engraftment of human blasts in patient-derived xenograft models of CRLF2-positive BCP-ALL. Importantly, givinostat killed ruxolitinib-resistant cells and potentiated the effect of current chemotherapy. Thus, givinostat in combination with conventional chemotherapy may represent an effective therapeutic option for these difficult–to-treat subsets of ALL. Lastly, the selective killing of cancer cells by givinostat may allow the design of reduced intensity regimens in CRLF2-rearranged Down syndrome-associated BCP-ALL patients with an overall benefit in terms of both toxicity and related complications.