Histone methylation and demethylation regulate B-cell development, and their deregulation correlates with tumor chemoresistance in diffuse large B-cell lymphoma, limiting cure rates. Since histone ...methylation status correlates with disease aggressiveness and relapse, we investigated the therapeutic potential of inhibiting histone 3 Lys27 demethylase KDM6B, in vitro, using the small molecule inhibitor GSK-J4. KDM6B is overexpressed in the germinal center B-cell subtype of diffuse large B-cell lymphoma, and higher KDM6B levels are associated with worse survival in patients with diffuse large B-cell lymphoma treated with R-CHOP. GSK-J4-induced apoptosis was observed in five (SU-DHL-6, OCI-Ly1, Toledo, OCI-Ly8, SU-DHL-8) out of nine germinal center B-cell diffuse large B-cell lymphoma cell lines. Treatment with GSK-J4 predominantly resulted in downregulation of B-cell receptor signaling and BCL6. Cell lines expressing high BCL6 levels or CREBBP/EP300 mutations were sensitive to GSK-J4. Our results suggest that B-cell receptor-dependent downregulation of BCL6 is responsible for GSK-J4-induced cytotoxicity. Furthermore, GSK-J4-mediated inhibition of KDM6B sensitizes germinal center B-cell diffuse large B-cell lymphoma cells to chemotherapy agents that are currently utilized in treatment regimens for diffuse large B-cell lymphoma.
We used clustered regularly interspaced short palindromic repeats/Cas9-mediated genomic modification to investigate B-cell receptor (BCR) signaling in cell lines of diffuse large B-cell lymphoma ...(DLBCL). Three manipulations that altered BCR genes without affecting surface BCR levels showed that BCR signaling differs between the germinal center B-cell (GCB) subtype, which is insensitive to Bruton tyrosine kinase inhibition by ibrutinib, and the activated B-cell (ABC) subtype. Replacing antigen-binding BCR regions had no effect on BCR signaling in GCB-DLBCL lines, reflecting this subtype's exclusive use of tonic BCR signaling. Conversely, Y188F mutation in the immunoreceptor tyrosine-based activation motif of CD79A inhibited tonic BCR signaling in GCB-DLBCL lines but did not affect their calcium flux after BCR cross-linking or the proliferation of otherwise-unmodified ABC-DLBCL lines. CD79A-GFP fusion showed BCR clustering or diffuse distribution, respectively, in lines of ABC and GCB subtypes. Tonic BCR signaling acts principally to activate AKT, and forced activation of AKT rescued GCB-DLBCL lines from knockout (KO) of the BCR or 2 mediators of tonic BCR signaling, SYK and CD19. The magnitude and importance of tonic BCR signaling to proliferation and size of GCB-DLBCL lines, shown by the effect of BCR KO, was highly variable; in contrast, pan-AKT KO was uniformly toxic. This discrepancy was explained by finding that BCR KO–induced changes in AKT activity (measured by gene expression, CXCR4 level, and a fluorescent reporter) correlated with changes in proliferation and with baseline BCR surface density. PTEN protein expression and BCR surface density may influence clinical response to therapeutic inhibition of tonic BCR signaling in DLBCL.
•The GCB subtype of DLBCL relies exclusively on tonic BCR signaling via CD79A Y188.•PTEN protein expression and BCR surface density determine the contribution of tonic BCR signaling to AKT activity in GCB-DLBCL.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Targeting BCL-2, a key regulator of survival in B-cell malignancies including precursor B-cell acute lymphoblastic leukemia, has become a promising treatment strategy. However, given the redundancy ...of anti-apoptotic BCL-2 family proteins (BCL-2, BCL-XL, MCL-1), single targeting may not be sufficient. When analyzing the effects of BH3-mimetics selectively targeting BCL-XL and MCL-1 alone or in combination with the BCL-2 inhibitor venetoclax, heterogeneous sensitivity to either of these inhibitors was found in ALL cell lines and in patient-derived xenografts. Interestingly, some venetoclax-resistant leukemias were sensitive to the MCL-1-selective antagonist S63845 and/or BCL-XL-selective A-1331852 suggesting functional mutual substitution. Consequently, co-inhibition of BCL-2 and MCL-1 or BCL-XL resulted in synergistic apoptosis induction. Functional analysis by BH3-profiling and analysis of protein complexes revealed that venetoclax-treated ALL cells are dependent on MCL-1 and BCL-XL, indicating that MCL-1 or BCL-XL provide an Achilles heel in BCL-2-inhibited cells. The effect of combining BCL-2 and MCL-1 inhibition by venetoclax and S63845 was evaluated in vivo and strongly enhanced anti-leukemia activity was found in a pre-clinical patient-derived xenograft model. Our study offers in-depth molecular analysis of mutual substitution of BCL-2 family proteins in acute lymphoblastic leukemia and provides targets for combination treatment in vivo and in ongoing clinical studies.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Pediatric acute myeloid leukemia (AML) is a highly heterogeneous disease making standardized measurable residual disease (MRD) assessment challenging. Currently, patient-specific DNA-based assays are ...only rarely applied for MRD assessment in pediatric AML. We tested whether quantification of genomic breakpoint-specific sequences via quantitative polymerase chain reaction (gDNA-PCR) provides a reliable means of MRD quantification in children with non-standardrisk AML and compared its results to those obtained with state-of-the-art ten-color flow cytometry (FCM). Breakpointspecific gDNA-PCR assays were established according to Euro-MRD consortium guidelines. FCM-MRD assessment was performed according to the European Leukemia Network guidelines with adaptations for pediatric AML. Of 77 consecutively recruited non-standard-risk pediatric AML cases, 49 (64%) carried a chromosomal translocation potentially suitable for MRD quantification. Genomic breakpoint analysis returned a specific DNA sequence in 100% (41/41) of the cases submitted for investigation. MRD levels were evaluated using gDNA-PCR in 243 follow-up samples from 36 patients, achieving a quantitative range of at least 10-4 in 231/243 (95%) of samples. Comparing gDNA-PCR with FCM-MRD data for 183 bone marrow follow-up samples at various therapy timepoints showed a high concordance of 90.2%, considering a cut-off of ≥0.1%. Both methodologies outperformed morphological assessment. We conclude that MRD monitoring by gDNA-PCR is feasible in pediatric AML with traceable genetic rearrangements and correlates well with FCM-MRD in the currently applied clinically relevant range, while being more sensitive below that. The methodology should be evaluated in larger cohorts to pave the way for clinical application.
Chromosome 21 is the most affected chromosome in childhood acute lymphoblastic leukemia. Many of its numerical and structural abnormalities define diagnostically and clinically important subgroups. ...To obtain an overview about their types and their approximate genetic subgroup-specific incidence and distribution, we performed cytogenetic, FISH and array analyses in a total of 578 ALL patients (including 26 with a constitutional trisomy 21). The latter is the preferred method to assess genome-wide large and fine-scale copy number abnormalities (CNA) together with their corresponding allele distribution patterns. We identified a total of 258 cases (49%) with chromosome 21-associated CNA, a number that is perhaps lower-than-expected because ETV6-RUNX1-positive cases (11%) were significantly underrepresented in this array-analyzed cohort. Our most interesting observations relate to hyperdiploid leukemias with tetra- and pentasomies of chromosome 21 that develop in constitutionally trisomic patients. Utilizing comparative short tandem repeat analyses, we were able to prove that switches in the array-derived allele patterns are in fact meiotic recombination sites, which only become evident in patients with inborn trisomies that result from a meiosis 1 error. The detailed analysis of such cases may eventually provide important clues about the respective maldistribution mechanisms and the operative relevance of chromosome 21-specific regions in hyperdiploid leukemias.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
While survival rates in paediatric acute lymphoblastic leukaemia (ALL) nowadays exceed 90%, systemic ALL relapse, especially after haemopoietic stem cell transplantation (HSCT), is associated with a ...poor outcome. As there is currently no standardized treatment for this situation, individualized treatment is often pursued. Exemplified by two clinical scenarios, the aim of this article is to highlight the challenge for treating physicians to find a customized treatment strategy integrating the role of conventional chemotherapy, immunotherapeutic approaches and second allogeneic HSCT. Case 1 describes a 2-year-old girl with an early isolated bone marrow relapse of an infant KMT2A-rearranged B-cell precursor ALL after allogeneic HSCT. After bridging chemotherapy and lymphodepleting chemotherapy, chimeric antigen receptor (CAR) T-cells (tisagenlecleucel) were administered for remission induction, followed by a second HSCT from the 9/10 human leukocyte antigen (HLA)-matched mother. Case 2 describes a 16-year-old girl with a late, isolated bone marrow relapse of B-cell precursor ALL after allogeneic HSCT who experienced severe treatment toxicities including stage IV renal insufficiency. After dose-reduced bridging chemotherapy, CAR T-cells (tisagenlecleucel) were administered for remission induction despite a CD19- clone without prior lymphodepletion due to enhanced persisting toxicity. This was followed by a second allogeneic HSCT from the haploidentical mother. While patient 2 relapsed around Day + 180 after the second HSCT, patient 1 is still in complete remission >360 days after the second HSCT. Both cases demonstrate the challenges associated with systemic ALL relapse after first allogeneic HSCT, including chemotherapy-resistant disease and persisting organ damage inflicted by previous therapy. Immunotherapeutic approaches, such as CAR T-cells, can induce remission and enable a second allogeneic HSCT. However, optimal therapy for systemic ALL relapse after first HSCT remains to be defined.
The mutational landscape of B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL), the most common pediatric cancer, is not fully described partially because commonly applied short‐read next ...generation sequencing has a limited ability to identify structural variations. By combining comprehensive analysis of structural variants (SVs), single‐nucleotide variants (SNVs), and small insertions‐deletions, new subtype‐defining and therapeutic targets may be detected. We analyzed the landscape of somatic alterations in 60 pediatric patients diagnosed with the most common BCP‐ALL subtypes, ETV6::RUNX1+ and classical hyperdiploid (HD), using conventional cytogenetics, single nucleotide polymorphism (SNP) array, whole exome sequencing (WES), and the novel optical genome mapping (OGM) technique. Ninety‐five percent of SVs detected by cytogenetics and SNP‐array were verified by OGM. OGM detected an additional 677 SVs not identified using the conventional methods, including (subclonal) IKZF1 deletions. Based on OGM, ETV6::RUNX1+ BCP‐ALL harbored 2.7 times more SVs than HD BCP‐ALL, mainly focal deletions. Besides SVs in known leukemia development genes (ETV6, PAX5, BTG1, CDKN2A), we identified 19 novel recurrently altered regions (in n ≥ 3) including 9p21.3 (FOCAD/HACD4), 8p11.21 (IKBKB), 1p34.3 (ZMYM1), 4q24 (MANBA), 8p23.1 (MSRA), and 10p14 (SFMBT2), as well as ETV6::RUNX1+ subtype‐specific SVs (12p13.1 (GPRC5A), 12q24.21 (MED13L), 18q11.2 (MIB1), 20q11.22 (NCOA6)). We detected 3 novel fusion genes (SFMBT2::DGKD, PDS5B::STAG2, and TDRD5::LPCAT2), for which the sequence and expression were validated by long‐read and whole transcriptome sequencing, respectively. OGM and WES identified double hits of SVs and SNVs (ETV6, BTG1, STAG2, MANBA, TBL1XR1, NSD2) in the same patient demonstrating the power of the combined approach to define the landscape of genomic alterations in BCP‐ALL.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Second malignant neoplasms (SMN) after primary childhood acute lymphoblastic leukemia (ALL) are rare. Among 1487 ALL patients diagnosed between 1981 and 2010 in Austria, the 10‐year cumulative ...incidence of an SMN was 1.1% ± 0.3%. There was no difference in the 10‐year incidence of SMNs with regard to diagnostic‐, response‐ and therapy‐related ALL characteristics except for a significantly higher incidence in patients with leukocytes ≥50.0 G/L at ALL diagnosis (2.1% ± 1.0% vs. 0% for 20.0–50.0 G/L, and 1.0% ± 0.3% for < 20.0 G/L; p = 0.033). Notably, there was no significant difference in the incidence of SMNs between patients with or without cranial radiotherapy (1.2% ± 0.5% vs. 0.8% ± 0.3%; p = 0.295). Future strategies must decrease the incidence of SMNs, as this event still leads to death in one‐third (7/19) of the patients.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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