Drivers Of Leukemia Research Articles

Clonal hematopoiesis and atherosclerosis.

Clonal hematopoiesis of indeterminate potential (CHIP) has emerged as a previously unrecognized, potent, age-related, and common risk factor for atherosclerosis. Somatic mutations in certain known leukemia driver genes give rise to clones of mutant cells in peripheral blood. The increased risk of developing hematologic malignancy does not, on its own, explain excess mortality in individuals with CHIP. Cardiovascular disease accounts for much of this gap. Experimental evidence supports the causality of certain CHIP mutations in accelerated atherosclerosis. CHIP due to mutations in different driver genes varies in their promotion of atherosclerotic events and in the region of augmented atherosclerotic involvement. For example, CHIP due to mutations in DNMT3a appears less atherogenic than CHIP that arises from TET2 or JAK2, forms of CHIP that incite inflammation. The recognition of certain CHIP mutations as promoters of atherosclerotic risk has opened new insights into understanding of the pathophysiology of this disease. The accentuated cardiovascular risk and involvement of distinct pathways of various forms of CHIP also inform novel approaches to allocation of targeted therapies, affording a step toward personalized medicine.

Early-life tobacco exposure is causally implicated in aberrant RAG-mediated recombination in childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children, yet few environmental risk factors have been identified. We previously found an association between early-life tobacco smoke exposure and frequency of somatic deletions of 8 leukemia driver genes among childhood ALL patients in the California Childhood Leukemia Study. To expand analysis genome-wide and examine potential mechanisms, we conducted tumor whole-genome sequencing in 35 ALL patients, including 18 with high prenatal tobacco exposure and 17 with low exposure as determined by established epigenetic biomarkers. High tobacco exposure patients had significantly more structural variants (P < .001) and deletions (P = .001) genome-wide than low exposure patients. Investigation of off-target RAG recombination revealed that 41% of deletions in the high tobacco exposure patients were putatively RAG-mediated (full RAG motif identified at one or both breakpoints) compared with only 21% in the low exposure group (P = .001). In a multilevel model, deletions in high tobacco exposure patients were 2.44-fold (95% CI:1.13-5.38) more likely to be putatively RAG-mediated than deletions in low exposure patients. No point mutational signatures were associated with prenatal tobacco exposure. Our findings suggest that early-life tobacco smoke exposure may promote leukemogenesis by driving development of somatic deletions in pre-leukemic lymphocytes via off-target RAG recombination.

Blockade of the lncRNA-DOT1L-LAMP5 axis enhances autophagy and promotes degradation of MLL fusion proteins.

Mixed-lineage leukemia (MLL) fusion gene caused by chromosomal rearrangement is a dominant oncogenic driver in leukemia. Due to having diverse MLL rearrangements and complex characteristics, MLL leukemia treated by currently available strategies is frequently associated with a poor outcome. Therefore, there is an urgent need to identify novel therapeutic targets for hematological malignancies with MLL rearrangements. qRT-PCR, western blot, and spearman correction analysis were used to validate the regulation of LAMP5-AS1 on LAMP5 expression. In vitro and in vivo experiments were conducted to assess the functional relevance of LAMP5-AS1 in MLL leukemia cell survival. We utilized chromatin isolation by RNA purification (ChIRP) assay, RNA pull-down assay, chromatin immunoprecipitation (ChIP), RNA fluorescence in situ hybridization (FISH), and immunofluorescence to elucidate the relationship among LAMP5-AS1, DOT1L, and the LAMP5 locus. Autophagy regulation by LAMP5-AS1 was evaluated through LC3B puncta, autolysosome observation via transmission electron microscopy (TEM), and mRFP-GFP-LC3 puncta in autophagic flux. The study shows the crucial role of LAMP5-AS1 in promoting MLL leukemia cell survival. LAMP5-AS1 acts as a novel autophagic suppressor, safeguarding MLL fusion proteins from autophagic degradation. Knocking down LAMP5-AS1 significantly induced apoptosis in MLL leukemia cell lines and primary cells and extended the survival of mice in vivo. Mechanistically, LAMP5-AS1 recruits the H3K79 histone methyltransferase DOT1L to LAMP5 locus, directly activating LAMP5 expression. Importantly, blockade of LAMP5-AS1-LAMP5 axis can represses MLL fusion proteins by enhancing their degradation. The findings underscore the significance of LAMP5-AS1 in MLL leukemia progression through the regulation of the autophagy pathway. Additionally, this study unveils the novel lncRNA-DOT1L-LAMP5 axis as promising therapeutic targets for degrading MLL fusion proteins.

Clonal Hematopoiesis Risk Score and All-Cause and Cardiovascular Mortality in Older Adults.

Clonal hematopoiesis (CH) with acquired pathogenic variants in myeloid leukemia driver genes is common in older adults but of unknown prognostic value. To investigate the prevalence of CH and the utility of the CH risk score (CHRS) in estimating all-cause and disease-specific mortality in older adults with CH. This population-based prospective cohort study involved community-dwelling older adults (aged 67-90 years) without hematologic malignant neoplasms (HMs) who were participants in the Atherosclerosis Risk in Communities Visit 5 at 4 US centers: Forsyth County, North Carolina; Jackson, Mississippi; Minneapolis, Minnesota; and Washington County, Maryland. Samples were collected from 2011 to 2013, sequencing was performed in 2022, and data analysis was completed in 2023. The exposure was a diagnosis of CH. CHRS scores (calculated using 8 demographic, complete blood cell count, and molecular factors) were used to categorize individuals with CH into low-risk (CHRS ≤9.5), intermediate-risk (CHRS >9.5 to <12.5), and high-risk (CHRS ≥12.5) groups. The primary outcome was all-cause mortality, and secondary outcomes were HM mortality, cardiovascular disease mortality, and death from other causes. Among 3871 participants without a history of HM (mean [SD] age, 75.7 [5.2] years; 2264 [58.5%] female individuals; 895 [23.1%] Black individuals; 2976 White individuals [76.9%]), 938 (24.2%) had CH. According to the CHRS, 562 (59.9%) were low risk, 318 (33.9%) were intermediate risk, and 58 (6.2%) were high risk. During a median (IQR) follow-up of 7.13 (5.63-7.78) years, 570 participants without CH (19.4%) and 254 participants with CH (27.1%) died. Mortality by CHRS risk group was 128 deaths (22.8%) for low risk, 93 (29.2%) for intermediate risk, and 33 (56.9%) for high risk. By use of multivariable competing risk regression, subdistribution hazard ratios (sHRs) for all-cause mortality were 1.08 (95% CI, 0.89-1.31; P = .42) for low-risk CH, 1.12 (95% CI, 0.89-1.41; P = .31) for intermediate-risk CH, and 2.52 (95% CI, 1.72-3.70; P < .001) for high-risk CH compared with no CH. Among individuals in the high-risk CH group, the sHR of death from HM (6 deaths [10.3%]) was 25.58 (95% CI, 7.55-86.71; P < .001) and that of cardiovascular death (12 deaths [20.7%]) was 2.91 (95% CI, 1.55-5.47; P < .001). In this cohort study, the CHRS was associated with all-cause, HM-related, and cardiovascular disease mortality in older adults with CH and may be useful in shared decision-making to guide clinical management and identify appropriate candidates for clinical trials.

Genomic landscape of patients with germline RUNX1 variants and familial platelet disorder with myeloid malignancy

AbstractFamilial platelet disorder with associated myeloid malignancies (FPDMM) is caused by germline RUNX1 mutations and characterized by thrombocytopenia and increased risk of hematologic malignancies. We recently launched a longitudinal natural history study for patients with FPDMM. Among 27 families with research genomic data by the end of 2021, 26 different germline RUNX1 variants were detected. Besides missense mutations enriched in Runt homology domain and loss-of-function mutations distributed throughout the gene, splice-region mutations and large deletions were detected in 6 and 7 families, respectively. In 25 of 51 (49%) patients without hematologic malignancy, somatic mutations were detected in at least 1 of the clonal hematopoiesis of indeterminate potential (CHIP) genes or acute myeloid leukemia (AML) driver genes. BCOR was the most frequently mutated gene (in 9 patients), and multiple BCOR mutations were identified in 4 patients. Mutations in 6 other CHIP- or AML-driver genes (TET2, DNMT3A, KRAS, LRP1B, IDH1, and KMT2C) were also found in ≥2 patients without hematologic malignancy. Moreover, 3 unrelated patients (1 with myeloid malignancy) carried somatic mutations in NFE2, which regulates erythroid and megakaryocytic differentiation. Sequential sequencing data from 19 patients demonstrated dynamic changes of somatic mutations over time, and stable clones were more frequently found in older adult patients. In summary, there are diverse types of germline RUNX1 mutations and high frequency of somatic mutations related to clonal hematopoiesis in patients with FPDMM. Monitoring changes in somatic mutations and clinical manifestations prospectively may reveal mechanisms for malignant progression and inform clinical management. This trial was registered at www.clinicaltrials.gov as #NCT03854318.

TRIP13 Is a Fetal-Enriched Therapeutic Target in NUP98-JARID1A + Pediatric Non-Down Syndrome AMKL

Chromosomal rearrangements have been identified as the main drivers of pediatric Acute Megakaryoblastic Leukemia in absence of Down syndrome (non-DS-AMKL). The t(11;12) involving NUP98 and KDM5A/JARID1A (NJ) accounts for approximately 15% of pediatric non-DS-AMKL cases and correlates with poor prognosis. An important aspect of AMKL is the strong enrichment in pediatric patients with frequent occurrence in infants (disease within 2 years post birth). In this study, we aimed to explore the role of cellular ontogeny in NJ-driven non-DS-AMKL and highlight TRIP13 as a fetal-enriched NJ-specific vulnerability acting through P53 regulation. To investigate the mechanism of NJ transformation, we first transduced murine hematopoietic stem and progenitor cells (HSPCs) originating from fetal liver (mFL-HSPCs) and bone marrow of adult mice (mBM-HSPCs) with a lentiviral vector overexpressing the cDNA encoding human NJ. In line with other studies, NJacts as a potent oncogene in vitro, sustaining cell proliferation and arresting cell differentiation. Both mFL-HSPCs and mBM-HSPCs expressing NJ transformed and dominated the culture after 3-4 weeks with an immature immune-phenotype (Lin-, Sca1+ and c-Kit+). Notably, outgrowth of mFL-HSPCs was significantly accelerated compared to adult mBM-HSPCs, despite similar transduction rates. Furthermore, in vivo experiments revealed that NJ-expressing mFL-HSPCs displayed a more aggressive phenotype (latency of 36 days) compared to mBM-HSPCs cells expressing NJ (latency of 70 days; P Long-rank &amp;lt; 0.001). Thus, both our in vitro and in vivo experiments strongly indicate an impact of fetal cell background. Suspecting an impact of fetal gene programs on NJ-mediated transformation, we performed a CRISPR-Cas9 screening targeting fetal expression signatures with a library probing 880 genes found to be deregulated when comparing fetal versus adult murine and human primary HSPCs. By comparing our findings with two other fetal liver-derived leukemia models - representing DS AMKL and familial platelet disorder with predisposition to AML (FPD-AML) -, we identified TRIP13 as a high-confidence candidate gene with exclusive dependency in NJ-driven non-DS-AMKL. Comparing human AML cell lines and NJ-overexpressing human fetal liver cells (hFL-HSPCs) confirmed selective targeting of NJ-driven cells by TRIP13 loss. To explore the molecular mechanism of TRIP13 sensitivity, we performed RNAseq. Surprisingly, gene set expression analysis (GSEA) revealed one highly significantly enriched pathway, i.e. TP53 signaling. Rescue experiments of Trip13 knockout in NJ-driven non-DS-AMKL cells further supported this finding: the massive depletion upon loss of Trip13 was completely reverted by either re-expression of a human TRIP13 cDNA or by knockout of Trp53. Of note, neither activation of TP53 signaling nor cell depletion was seen in healthy hematopoietic cells from FL upon Trip13 perturbation. Finally, we aimed to leverage TRIP13 dependency therapeutically. To this end, we first explored Trip13-depletion in an in vivo setting. In a fluorescence-based competitive transplantation assay, Trip13-ablated leukemic blasts were significantly diminished in the bone marrow of recipient mice after 4 weeks, showing a mean depletion of 83%. Next, NJ-driven non-DS-AMKL mFL-HSPCs were treated with the TRIP13 inhibitor DCZ0415. Similarly to genetic TRIP13 ablation, DCZ0415-treated NJ mFL-HSPCs showed a significantly higher sensitivity to the drug compared to models of DS AMKL, FPD-AML, and healthy HSPCs with fold changes of 1.6 (p-value = 0.024), 2.3 (p-value = 0.001) and 2.0 (p-value = 0.008), respectively. To exploit our mechanistic knowledge on TRIP13 ablation-mediated P53 activation, we further combined DCZ0415 with the MDM2 inhibitor Idasanutlin. In line with our mechanistic data, a high synergy (Bliss synergy score = 9.7) of TRIP13 and MDM2 inhibition was observed. In conclusion, our study uncovers TRIP13 as a fetal-enriched vulnerability in NJ-driven non-DS-AMKL, mechanistically acting through P53, which we leveraged for a mechanism-driven treatment approach with dual TRIP13/MDM2 inhibition as a potential therapeutic strategy for the treatment of high-risk pediatric non-DS-AMKL.

Synergy of Stag2-Cohesin Loss Results in Expansion of Npm1c-Mutant Hematopoietic Stem and Progenitor Cells

STAG2 is a member of cohesin complex that is recurrently mutated in &amp;gt;10 cancers and is essential in maintaining the integrity of the 3D genome partitioning structure known as topologically structural domains (TADs). Our previous work has demonstrated that depletion of various cohesin factors, including Stag2, leads to increased hematopoietic stem and progenitor population (HSPC) self-renewal and myeloid-biased differentiation. Loss of Stag2 leads to impaired sub-TADs and affects key hematopoietic transcription factors, such as PU.1, to access and engage their target genes. Yet, the extent and cooperative transformational role of Stag2-cohesin with leukemia driver mutations, such as Npm1c, remains unexplored. To determine whether Stag2-cohesin regulates leukemic 3D chromatin organization, we generated dual Stag2 Δ Npm1 c/+ murine models with tamoxifen inducible UbcCreER T2. After 4 weeks, HSPCs (LSK cells; Lin-Sca1+Kit+) are increased in Stag2 Δ Npm1 c/+ double mutant mice ( Figure 1A). Within LSK, Stag2 Δ Npm1 c/+ but not Npm1 c/+ has marked expansion of the myeloid bias MPP2 (LSK+Flk2-Cd150+Cd48+) and MPP3 (LSK+Flk2-Cd150-Cd48+) compartment. Interestingly, Stag2 Δ single mutant exhibit LSK and MPP3 expansion at 16 weeks, suggesting the early expansion of HSPC compartment is the result of Stag2 ΔNpm1 c/+ synergistic acceleration. No difference in serial replating of LSK and GMP (LSK-Cd34+FcyRII+) cells were observed between Npm1 c/+ and Stag2 Δ Npm1 c/+. However, Stag2 Δ Npm1 c/+ LSK cells had impaired reconstitution capacity and myeloid biased output in primary and secondary transplantation. To determine if the blockage is specific to Stag2-cohesin loss, we generated the Smc3/Npm1c double mutant mice, where both Stag1-cohesin and Stag2-cohesin complexes were partially depleted upon heterozygous deletion of Smc3. Interestingly, there were no changes in LSK or MPP3 population in Smc3 +/- Npm1 c/+ mice, suggesting it is specific to Stag2-cohesin loss which obstructs differentiation in Npm1 c/+ HSPC. Mechanistically, we performed bulk ATAC-seq on sorted HSC LT (LSK+Flk2-Cd150+Cd48-) and MPP3 population, as poor long-term reconstitution suggests functional alteration in HSC LT. Comparing to WT, both Stag2 Δ and Stag2 Δ Npm1 c/+ HSC LT have increased chromatin accessibility, while only Stag2 Δ Npm1 c/+ MPP3 have persistent opening of chromatin. Motif analysis of increased accessible peaks identified RUNX2, GATA3 and MEIS1 are among the increased accessible region in Stag2 Δ Npm1 c/+ MPP3, which reflects the increased myeloid output in the transplantation. Recently, various group have suggested that Npm1c directly binds to chromatin and drives the overexpression of genes, especially at HOX clusters and MEIS1. This suggests a possible synergistic mechanism which Stag2-cohesin regulates chromatin accessibility of key TF loci, which influences normal and leukemic stem and progenitor differentiation. To comprehensively profile the LSK population, we performed scRNAseq at 4 weeks post mutation activation, results showed a marked immune pathway activation in Stag2 Δ Npm1 c/+ LSK cells. To inter cell fate transition, we employed CellDancer, an RNA velocity based algorithm and found that Stag2 Δ Npm1 c/+ MPP cells have altered differentiation trajectory and delayed kinetics at MPP3 stages, suggesting a differentiation block ( Figure 1B). In conjunction with our results, we postulate that Stag2-cohesin regulates TF interactions with regulatory elements during cell fate transitions. We are currently investigating the direct interaction of Stag2-cohesin with regulatory elements in Npm1 c/+ cells and compare to WT and Stag2 Δ Npm1 c/+ cells. We will investigate the 3D genome organization of Npm1 c/+ and Stag2 Δ Npm1 c/+ cells to determine whether Stag2 loss induces the phenotypic changes via Ctcf-dependent or independent manner.

Clonal Hematopoiesis Is Common in Unrelated Stem Cell Donors but Has No Impact on Patient Outcome after Hematopoietic Stem Cell Transplantation

Introduction. Clonal hematopoiesis (CH) has been associated with increased mortality mainly due to associations with cardiovascular diseases and hematologic cancer. Previous studies with predominantly related stem cell donors suggest that donor CH modulates graft-versus-host reactions and may augment graft-versus-leukemia effects after allogeneic hematopoietic cell transplantation (alloHCT). The impact of donor CH in the setting of unrelated alloHCT remains to be determined. To address this question, we initiated a joint study of the Transplant Complications Working Party of EBMT and DKMS. Patients and Methods. Donor samples from the time of stem cell donation were taken from the Collaborative Biobank. Patient baseline and outcome data were retrieved from the EBMT registry. Unrelated donor-recipient pairs were selected for a minimum donor age of 35 years. CH analysis was performed with an error-corrected ultra-deep NGS assay for 45 genes with a median sensitivity of 0.2% variant allele frequency (VAF). Accuracy of low frequency CH findings was checked by cross validation with an orthogonal assay. The primary objective was to assess the impact of donor CH on overall survival (OS). Major secondary endpoints were the risk of relapse/progression and non-relapse mortality (NRM), cumulative incidences of acute and chronic GVHD and event-free survival (EFS). All endpoints were evaluated with (cause-specific) multivariable Cox regression models. Adjustment factors were patient and donor age, diagnosis, disease risk index, Karnofsky performance status, conditioning intensity, GVHD-prophylaxis, graft source, HLA-match, sex match and CMV match. We did not adjust for multiple testing. Results. Data from 2584 unrelated donor-recipient pairs were analyzed. Median patient follow-up after alloHCT was 60 months. The median donor age was 46 years (range, 39-61 years). The median patient age was 54 years (range, 0-79 years). Indications for alloHCT were AML (44%), ALL (10%), MDS (10%), MPN (6%), B-cell lymphoma (6%), Multiple Myeloma (5%), CML 3%, inherited disorders (3%), CLL 2% and other (13%). Transplantations were performed between 2005 and 2018. In vivo or ex vivo T-cell depletion (TCD) was used in 81% of transplants, PTCY in 5% and no TCD in 14%. Mutations in DNMT3A, TET2, and ASXL1 defined CH most frequently. The distribution of donor CH across leukemia driver genes was comparable to previous reports (Panel A). With 0.2% (2%) VAF cutoffs, rates of DNMT3A-mutations were 19% (2%) among donors younger than 41 years compared to 37% (6%) among donors aged 55 years and higher. Corresponding rates for 0.2% (2%) VAF cutoffs for total CH were 38% (4%) among donors younger than 41 years compared to 67% (14%) among donors aged 55 years and higher. We tested systematically increasing VAF cutoffs (≥0.2% versus &amp;lt;0.2%, ≥1% versus &amp;lt;1%, ≥2% versus &amp;lt;2%, ≥5% versus &amp;lt;5%, and ≥5% versus &amp;lt;0.2%) for DNMT3A-CH and total CH for associations with clinical endpoints. Panel B shows results for multivariable regression analyses for total donor CH. Although we observed trends towards increased mortality and inferior EFS for some cutoffs, no incremental impact of donor CH with higher VAF was found and no systematic changes of the risk for GVHD and relapse/progression were observed. Conclusions. We conclude that donor CH has no significant impact on the risk for GVHD, relapse and survival in the context of HLA-compatible unrelated donor alloHCT with GVHD-prophylaxis based predominantly on T-cell depletion with ATG.

Clonal Medicine Targeting DNA Damage Response Eradicates AML

Clonal diversity of acute myeloid leukemia (AML) plays a key role in poor therapeutic outcomes. AML cells usually accumulate spontaneous DNA damage including highly toxic DNA double-strand breaks (DSBs) induced by metabolic products and replication stress. To repair numerous DSBs and survive, AML cells activate the DNA damage response (DDR) involving the pathways that sense (ATM and ATR kinases) and repair (RAD51-mediated homologous recombination = HR, RAD52-mediated transcription associated homologous recombination = TA-HR and single strand annealing = SSA, DNA-PK -mediated non-homologous end-joining = NHEJ, PARP1/Polq-dependent microhomology-mediated end-joining = MMEJ) DSBs. Thus, DDR is a legitimate therapeutic target. Numerous AML-driving mutations [ FLT3(ITD), TETmut, DNMT3Amut, AML1-ETO, PML-RARa , IDH1mut, cKITmut] regulate DDR and affect the sensitivity of leukemia cells to DDR inhibitors (DDRi). The genetic landscape of malignant clones in a patient may be complicated since individual clones can carry multiple mutations. Thus, individual AML clones may respond differently to DDRi depending on their mutational profile. In our experimental protocol AML cells from individual patients were treated with various DDRi followed by single-cell targeted DNA sequencing (sctDNA-seq) to integrate patient's leukemia clonal composition with response to the inhibitors. We developed a sctDNA-seq myeloid platform interrogating up to 1394 genetic variants of 54 known leukemia driver genes, which unraveled the clonal landscape of AML at a single-cell resolution before and after the treatment. Based on these results we designed a “clonal attack”, a patient-tailored combination of DDRi targeting all AML clones in a patient sample. Here we show that DDRi simultaneously attacking different clones caused clonal attrition and eradicated the disease in vitro and in vivo. For example, in AML-MD2 sample 2 clones carrying EZH2(V679M) + TET2(L1721W) and EZH2(V679M) + TET2(L1721W) + FLT3(D835Y) were resistant/less responsive to ATRi but sensitive to PARPi, ATMi and RAD52i. Conversely, three other clones carrying EZH2(V679M) + TET2(L1721W) + RUNX1(D160Y), EZH2(V679M) + TET2(L1721W) + RUNX1(D160Y) + EZH2(E54*) and EZH2(V679M) + TET2(L1721W) + RUNX1(D160Y)EZH2(E54*) + BCOR1(R1334Tfs*32) + NRAS(G13R) were sensitive to ATRi while less responsive to PARPi, ATMi and RAD52i. Based on this observation, we hypothesized that simultaneous treatment with ATRi + PARPi or ATRi + ATMi should result in elimination of all AML-MD2 clones. In agreement with this hypothesis, in vitro treatment with these combinations was 20 - 30x more effective in eliminating clonogenic growth of Lin-CD34+ AML-MD2 cells when compared to individual inhibitors. This effect was associated with abundant accumulation of DSBs. Combinations of these inhibitors were only modestly toxic to normal hematopoietic cells. Moreover, combination of DDRi displaying similar clonal targeting specificity was only 2x better than individual inhibitors. Next, humanized NRGS immunodeficient mice bearing primary AML-MD2 xenograft were treated with vehicle, PARPi, ATRi and the combination of these drugs. We found that individual treatments reduced the number of hCD45+ AML-MD2 cells in bone marrow and spleen by approximately 2x and 3x, respectively. Remarkably, the combination of PARPi + ATRi eliminated more than 99% of leukemia cells in bone marrows of 7/10 mice and in spleens of 10/10 mice while no obvious toxicity was observed. Clonal targeting by DDR inhibitors, however, may not be applicable to all AML samples. For example, we identified AML patient samples, where clones displayed similar sensitivity pattern or were resistant to DDRi treatment. In conclusion, we postulate that sctDNA-seq combined with in vitro DDRi sensitivity testing (sctDNA-seq/DDRi) is a powerful tool to interrogate clonal sensitivity of AML to these agents. This clonal medicine approach may become a novel therapeutic regimen to overcome clonal complexity of AML in a cohort of patients. The “clonal attack” by DDR inhibitors shifts the paradigm of genotoxic therapies from those using non-discriminative cytotoxic drugs to those selectively attacking DDR vulnerabilities in AML clones with minimal harm to normal cells. Since clonal heterogeneity and DNA damage are hallmarks of cancer, the “clonal attack” may be broadly applicable to the quest for cancer cure.

Exploration of rotational thromboelastometry (ROTEM) to characterize the coagulation profiles of newly diagnosed pediatric leukemia patients

BackgroundViscoelastic testing has been used in adult hematologic malignancies in conjunction with conventional coagulation tests (CCTs) to predict coagulopathies and tailor blood product replacement. However, there is a paucity of similar pediatric studies. ObjectivesAnalyze and correlate leukemia-associated coagulopathy in newly diagnosed pediatric leukemia patients using CCT’s and Rotational Thromboelastometry (ROTEM). MethodsPediatric patients with newly diagnosed acute leukemia underwent testing with ROTEM and CCTs on days 0, 15 and 29 of induction chemotherapy. ResultsSixty-two patients were enrolled. At presentation, 54.8 % of patients had platelets <50 K/μL, 73 % had prolonged PT, 1.6 % had fibrinogen <150 mg/dL. Fifteen patients (24.2 %) had WHO grade 1 bleeding and two patients (3 %) had WHO grade 4 bleeding. EXTEM/INTEM values at presentation (day 0) reflected hypocoagulability, however FIBTEM revealed hypercoagulability. Patients showed a progressive hypocoagulability in all ROTEM assays by day 15 (day 0 vs day 15, p < 0.001), with improvement by day 29 (day 15 vs day 29, p < 0.001). Day 0 ROTEM parameters were comparable to day 29. Fibrinogen strongly correlated with ROTEM at all three time points (p < 0.0001), along with platelet count with moderate correlations (p < 0.001). ConclusionFibrinogen and platelets appear to be the drivers of leukemia associated coagulopathy in the pediatric population, suggesting the utility of using CCTs and ROTEM in this population to better evaluate hemostatic function and guide blood product replacement.

Clonal Targeting of DNA Damage Response Pathways Eradicates Myeloproliferative Neoplasms

Clonal diversity of myeloproliferative neoplasms (MPN) plays a key role in poor therapeutic outcomes. MPN cells usually accumulate spontaneous DNA damage including highly toxic DNA double-strand breaks (DSBs) induced by metabolic products and replication stress. To repair numerous DSBs and survive, MPN cells activate the DNA damage response (DDR) involving the pathways that sense (ATM and ATR kinases) and repair (RAD51-mediated homologous recombination = HR, RAD52-mediated transcription associated homologous recombination = TA-HR and single strand annealing = SSA, DNA-PK -mediated non-homologous end-joining = NHEJ, PARP1/Polq-dependent microhomology-mediated end-joining = MMEJ) DSBs. Thus, DDR is a legitimate therapeutic target. Numerous MPN-driving mutations [ JAK2(V617F), TETmut, DNMT3Amut, IDH1mut] regulate DDR and affect the sensitivity of leukemia cells to DDR inhibitors (DDRi). The genetic landscape of malignant clones in a patient may be complicated since individual clones can carry multiple mutations. Thus, individual MPN clones may respond differently to DDRi depending on their mutational profile. In our experimental protocol MPN cells from individual patients were treated with various DDRi followed by single-cell targeted DNA sequencing (sctDNA-seq) to integrate patient's leukemia clonal composition with response to the inhibitors. We developed a sctDNA-seq myeloid platform interrogating up to 1394 genetic variants of 54 known leukemia driver genes, which unraveled the clonal landscape of MPN at a single-cell resolution before and after the treatment. Based on these results we designed a “clonal attack”, a patient-tailored combination of DDRi targeting all MPN clones in a patient sample. Here we show that DDRi simultaneously attacking different clones caused MPN clonal attrition in vitro. For example, phylogenetic tree analysis of MPN patient P349 sample detected linear multi-clonal architecture with 3 Lin-CD34+ clones carrying specific sets of mutations. sctDNA-seq followed by fish plot analysis revealed clonal similarities and differences in response to DDRi. The clone carrying KMT2A(L2373H) + SETBP1(H1100R) was more sensitive to ATRi than RAD52i, conversely clones with KMT2A(L2373H) alone and KMT2A(L2373H) + SETBP1(H1100R) + FLT3(R834L) were more sensitive to RAD52i than ATRi. Based on this observation, we hypothesized that simultaneous treatment with ATRi+RAD52i should result in elimination of all 3 MPN P349 clones. Remarkably, the combination of RAD52i + ATRi was &amp;gt;100x more effective in inhibiting clonogenic growth of Lin-CD34+ P349 cells (all clonogenic cells were eradicated). On the other hand, combination of RAD52i + ATMi, the two DDR inhibitors displaying similar pattern of clonal targeting was only 2x better than individual inhibitors. Phylogenetic tree analysis of Lin-CD34+ cells from another MPN patient P350 sample showed linear multi-clonal architecture with 4 clones. Again, sctDNA-seq followed by fish plot analysis revealed clonal similarities and differences in response to DDRi. All 4 clones displayed similar sensitivity to RAD52i and ATMi, but they responded differently to PARPi and ATRi. Clones carrying TET2(P363L) + NRAS(G12D) were more sensitive to ATRi than PARPi, whereas clones with TET2(P363L) + NRAS(G12D) + DNMT3A(W330C) and TET2(P363L) + NRAS(G12D) + DNMT3A(W330C) + IDH2(R132C) responded better to PARPi than ATRi. Importantly, the combination of PARPi + ATRi was &amp;gt;9x more effective in inhibiting clonogenic growth of Lin-CD34+ P349 cells, whereas RAD52i + ATMi was only 2x better than individual inhibitors. In conclusion, we postulate that sctDNA-seq combined with in vitro DDRi sensitivity testing (sctDNA-seq/DDRi) is a powerful tool to interrogate clonal sensitivity of MPN to these agents. This clonal medicine approach may become a novel therapeutic regimen to overcome clonal complexity of MPN in a cohort of patients. The “clonal attack” by DDR inhibitors shifts the paradigm of genotoxic therapies from those using non-discriminative cytotoxic drugs to those selectively attacking DDR vulnerabilities in MPN clones with minimal harm to normal cells. Since clonal heterogeneity and DNA damage are hallmarks of cancer, the “clonal attack” may be broadly applicable to the quest for cancer cure.

Uncovering NOTCH1 as a Promising Target in the Treatment of MLL-Rearranged Leukemia.

MLL rearrangement (MLLr) is responsible for the development of acute leukemias with poor outcomes. Therefore, new therapeutic approaches are urgently needed. The NOTCH1 pathway plays a critical role in the pathogenesis of many cancers including acute leukemia. Using a CRISPR/Cas9 MLL-AF4/-AF9 translocation model, the newly developed NOTCH1 inhibitor CAD204520 with less toxic side effects allowed us to unravel the impact of NOTCH1 as a pathogenic driver and potential therapeutic target in MLLr leukemia. RNA sequencing (RNA-seq) and RT-qPCR of our MLLr model and MLLr cell lines showed the NOTCH1 pathway was overexpressed and activated. Strikingly, we confirmed this elevated expression level in leukemia patients. We also demonstrated that CAD204520 treatment of MLLr cells significantly reduces NOTCH1 and its target genes as well as NOTCH1 receptor expression. This was not observed with a comparable cytarabine treatment, indicating the specificity of the small molecule. Accordingly, treatment with CAD204520 resulted in dose-dependent reduced proliferation and viability, increased apoptosis, and the induction of cell cycle arrest via the downregulation of MLL and NOTCH1 target genes. In conclusion, our findings uncover the oncogenic relevance of the NOTCH1 pathway in MLLr leukemia. Its inhibition leads to specific anti-leukemic effects and paves the way for further evaluation in clinical settings.

Diagnostic utility of whole genome sequencing in adults with B-other acute lymphoblastic leukemia

AbstractGenomic profiling during the diagnosis of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) in adults is used to guide disease classification, risk stratification, and treatment decisions. Patients for whom diagnostic screening fails to identify disease-defining or risk-stratifying lesions are classified as having B-other ALL. We screened a cohort of 652 BCP-ALL cases enrolled in UKALL14 to identify and perform whole genome sequencing (WGS) of paired tumor-normal samples. For 52 patients with B-other, we compared the WGS findings with data from clinical and research cytogenetics. WGS identified a cancer-associated event in 51 of 52 patients, including an established subtype defining genetic alterations that were previously missed with standard-of-care (SoC) genetics in 5 of them. Of the 47 true B-other ALL, we identified a recurrent driver in 87% (41). A complex karyotype via cytogenetics emerges as a heterogeneous group, including distinct genetic alterations associated with either favorable (DUX4-r) or poor outcomes (MEF2D-r and IGK::BCL2). For a subset of 31 cases, we integrated the findings from RNA sequencing (RNA-seq) analysis to include fusion gene detection and classification based on gene expression. Compared with RNA-seq, WGS was sufficient to detect and resolve recurrent genetic subtypes; however, RNA-seq can provide orthogonal validation of findings. In conclusion, we demonstrated that WGS can identify clinically relevant genetic abnormalities missed with SoC testing as well as identify leukemia driver events in virtually all cases of B-other ALL.

DNTT activation, TdT-aided gene length mutation, and better prognosis in ATG-based regimen allo-HSCT in AML.

This study aimed to investigate the relationship between anomalous DNA nucleotidylexotransferase (DNTT) activation and the mutagenesis of gene length mutations (LMs) in acute myeloid leukemia (AML), and the relevance of their prognosis in antithymocyte globulin (ATG)-based regimen allogeneic hematopoietic stem cell transplantation (allo-HSCT). A cohort of 578 AML cases was enrolled. Next-generation sequencing was performed to screen mutations of 86 leukemia driver genes. RNA-seq was used to analyze gene expression. Prognostic analysis was investigated in 239 AML cases who underwent ATG-based regimen allo-HSCT. We report a refined subtyping algorithm of LMs (type I-IV) based on sequence anatomy considering the TdT-aided mutagenesis mechanism. GC content adjacent to LM junctions, inserted nontemplate nucleotide bases, and DNTT expression analysis supported the DNTT activation and TdT-aided mutagenesis in type II/III LMs in the total AML cohort. Both single-variate and multivariate analyses showed a better overall survival of FLT3 type III compared to type I in a subset of ATG-based regimen allo-HSCT cases. The novel LM subtyping algorithm not only deciphers the etiology of the mutagenesis of LMs but also helps to fine-tune prognosis differentiation in AML. The possible prognostic versatility of this novel LM subtyping algorithm in terms of chemotherapy, targeted therapy, and allo-HSCT merits further investigation.

Abstract P011: Clonal hematopoiesis and risk of lethal prostate cancer: a prospective cohort study with long-term follow-up

Abstract Background: Clonal hematopoiesis (CH), the presence of acquired mutations in leukemia driver genes, promotes systemic inflammation and is common among aging men. Prostate cancer, a subset of which is lethal, also develops in aging men. We hypothesized that CH contributes to development of lethal prostate cancer. Methods: We conducted nested case-cohort studies for metastatic prostate cancer and prostate cancer-specific death (lethal prostate cancer) within the prospective Health Professionals Follow-up Study. First, we followed 1155 men free of prostate cancer and cardiovascular disease at blood draw (1993-1995) for development of lethal prostate cancer over up to 26 years. Second, we followed 532 men with incident non-metastatic prostate cancer for development of lethal prostate cancer. We sequenced blood DNA from 1488 participants for putative CH driver mutations in the 9 most common CH-defining genes with a custom targeted panel (VariantPlex, Invitae, Inc.) at ultra-high depth (mean, 18,000x), employing unique molecular identifiers for error correction. CH variant calling used a novel ensemble calling approach, ArCCH, validated with in-silico tumor dilutions and blinded technical replicates, which had high accuracy for variant allele frequencies (VAFs) as low as 0.1%. We estimated hazard ratios (HRs) with 95% confidence intervals (CIs) in proportional hazards regression with Prentice case-cohort weights. Results: In a random sample of 968 men initially free of prostate cancer and cardiovascular disease (median age at blood draw 60 years, interquartile range 52 to 67), 80% of men had CH at a variant allele frequency (VAF) of &amp;gt;0.1%, 15% had VAFs &amp;gt;2%, and 3% had VAFs &amp;gt;10%; 75% of men had variants in epigenetic modifier genes (DNMT3A, TET2, ASXL1), and 21% had variants in DNA repair genes (PPM1D, TP53, CHEK2). CH burden was strongly age-associated, with approximately one additional CH variant per decade of age at blood draw (mean difference 1.07 variants, 95% CI 0.93 to 1.21). Among men initially free from prostate cancer, after adjusting for age at blood draw, CH clones between 0.1% and 10% VAF were not related to lethal prostate cancer (206 events total; HR 0.93, 95% CI 0.49-1.76 for VAFs 2-10% vs. no variants detected at &amp;gt;0.1% VAF). Results for epigenetic modifiers and DNA repair genes were similar. While inconclusive, data were compatible with positive associations among younger men (&amp;lt; 65 years) or for VAFs &amp;gt;10%. Among men initially diagnosed with non-metastatic prostate cancer, results were similarly null for progression to lethal prostate cancer (164 events). Conclusions: This large prospective study with long-term follow-up suggests that low-level CH is unlikely a major contributor to and not well suited for early detection of lethal prostate cancer. Citation Format: Konrad H. Stopsack, Irenaeus C. Chan, Evelyn Schmidt, Alex Panchot, Samantha McNulty, Nicole A. Schreiber, Yiwen Zhang, Kathryn L. Penney, Michael F. Berger, Luis A. Diaz, Ross L. Levine, Kelly L. Bolton, Lorelei A. Mucci, Philip W. Kantoff. Clonal hematopoiesis and risk of lethal prostate cancer: a prospective cohort study with long-term follow-up. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P011.

Germline SAMD9/9L MDS Predisposition Syndromes Are Characterized By Complex Clonal Architecture and Lineage-Specific Escape Mechanisms Including Somatic Genetic Rescue in T and B Lymphocytes

Inherited bone marrow failure (BMF) and MDS predisposition syndromes have an intrinsic defect resulting in loss and/or dysfunction of hematopoietic stem and progenitor cells (HSPC). HSPC clones can escape this process by acquiring gene- or genome-level genetic changes (compensatory mutations or uniparental disomies (UPD)) thereby increasing cellular fitness. This process, referred to as somatic genetic rescue (SGR) has been shown to occur in >70% of patients with gain-of-function mutations in SAMD9 and SAMD9L (SAMD9/9L) genes, which are a common cause of early onset BMF and pediatric MDS with monosomy 7. It is unknown how SGR arises and in which cells it originates. Furthermore, little is known about the epistatic interactions of various rescue events. We aimed to address these questions by utilizing a newly established single cell (sc)-proteogenomic platform that can simultaneously detect single nucleotide variants (SNVs) and copy number variation (CNVs) in addition to protein expression. We applied this technology to study clonal architecture and cellular origins of SGR lesions in a cohort of clinically annotated SAMD9/9L-mutated (SAMD9/9Lmut) patients. We also asked if monosomy 7, traditionally assessed by karyotyping, can be also detected in peripheral blood (PB) of affected patients. We first performed whole exome and panel sequencing to characterize 67 germline SAMD9/9Lmut patients. Guided by this data, we designed a sc-proteogenomic panel with 69 amplicons specific to variants in SAMD9/9L and leukemia driver genes, and 250 amplicons covering high-frequency SNVs on chromosome 7q. For validation of initial findings, we developed a second panel targeting full coding regions of SAMD9/9L genes, and additional 7p SNVs allowing for a precise delineation of uniparental isodisomy 7q (UPD7q). We included oligonucleotide-conjugated antibodies specific to major hematopoietic antigens, including CD19, CD3, CD11b, CD38 and CD34. Using this panel, we analyzed 22 samples (15 bone marrow (BM) and 7 PB) of 10 patients enrolled in local biobanking protocols. On average, 3,845 high-quality cells were genotyped per sample, and we robustly identified SNVs, monosomy 7 and UPD7q changes in single cells. Somatic rescue SAMD9/9Lmut were found only in clones with normal (diploid) chromosome 7, UPD7q (containing two wild type SAMD9/9L alleles after mitotic recombination event) was a stand-alone clone, while monosomy 7 (SAMD9/9Lmut lost) occurred alone or together with leukemia driver mutations. We also detected lesions that were previously missed using routine clinical sequencing in 3 patients (somatic SAMD9/9Lmut or UPD7q). Most patients had branching clonal architecture (i.e., independent monosomy 7, UPD7q, and somatic SAMD9/9Lmut), while linear trajectories were rare (i.e., monosomy 7 à + leukemia driver mutation). Serial assessment of 4 patients with stable hematology (3 with monosomy 7 and 1 with an additional somatic SAMD9/9Lmut) for a period of up to 4.8 years, revealed stable clonal populations without acquisition of additional driver mutations. Quantification of clones in paired BM and PB in 4 patients demonstrated equal frequencies of monosomy 7 and somatic SAMD9/9Lmut in both compartments, demonstrating the utility of this assay in surveillance. Our findings were confirmed by sorting BM cells into different lineages followed by sequencing, error-corrected sequencing, and ddPCR. Finally, analysis of protein expression data revealed that while monosomy 7 is present exclusively in cells with myeloid phenotype, the SGR events can arise in lymphocyte lineage only; 4 patients carried clones with somatic SAMD9/9Lmut in CD3+ lymphocytes, and 2 patients had UPD7q clones in either CD3+ or CD19+ lymphocytes. These findings provide clues why most of the SGR events in patients do not result in long-lasting clinical remission since the genetic rescue can be restricted only to certain hematopoietic lineages. In conclusion, single cell proteogenomic analysis is useful for the understanding the mechanisms of clonal evolution in SAMD9/9L syndromes. This method also offers the opportunity for less invasive surveillance of karyotype abnormalities from peripheral blood in patients who are at high risk for MDS progression.

Uncovering the Enemy: Single-Cell Transcriptional Profiling of Measurable Residual Disease (MRD) Cells

Albeit novel treatment concepts have entered the therapeutic landscape of AML, relapse rates remain high. This is attributed to treatment-refractory leukemic cells, which can be quantified by flow cytometry and molecular techniques (Measurable Residual Disease, MRD). MRD positivity is a significant prognostic biomarker for relapse and low overall survival rates. Post-remission therapy for the elimination of MRD remains a major challenge. We previously confirmed the prognostic value of early flow MRD assessment during aplasia in patients who achieve complete remission after intensive induction therapy (Koehnke et al., 2019). Here, we hypothesized that single-cell transcriptional profiling of refractory leukemic cells using flow cytometry (flow MRD) during aplasia will help to understand resistance mechanisms to therapy and to identify suitable targets for cellular immunotherapy. Flow MRD detection is based on the definition and characterization of leukemia-associated aberrant immunophenotypes (LAIPs) at the time of initial diagnosis, as described previously (Koehnke et al., 2015). A similar approach was applied to define LAIP+ cells at the time of aplasia (d16-d21 after initiation of induction therapy). For single-cell RNA-seq, LAIP+ cells from paired initial diagnosis and aplasia were sorted and subjected to transcriptional profiling using the mcSCRB-seq (molecular crowding single-cell RNA barcoding and sequencing) protocol (Bagnoli et al., 2018). We included 7 AML patients of different ELN genetic risk group (favorable, n=1; intermediate, n=4; adverse, n=2). In total, 854 LAIP+ cells from paired initial diagnosis-aplasia samples were projected using uniform manifold approximation and projection (UMAP) visualizations. We observed a distinct clustering of LAIP+ cells per patient and time point, revealing high inter-patient heterogeneity (Figure 1A). To identify common transcriptional changes across patients, we performed differential gene expression analysis of LAIP+ cells, comparing aplasia to initial diagnosis. We identified 93 genes significantly upregulated and 41 genes significantly downregulated (p < 0.01, FC >1 or <-1; Figure 1B). Among markedly upregulated genes across patients at the time of aplasia, we found 3 genes related to leukemic cell maintenance. Interestingly, SMARCA4, an ATPase subunit of the SWI/SNF chromatin remodeling complex, known to be essential for the proliferation and survival of AML cells (Buscarlet et al., 2014; Shi et al., 2013), showed a 1.99-fold increase in expression (p < 0.00). In addition, TRAP1, a candidate target in cancers with antiapoptotic function, was upregulated 1.5-fold (p < 0.00). And lastly, LIN28B, the cancer stem cell and leukemia driver gene, was also found to be upregulated by 0.7-fold in aplasia LAIP+ cells (p < 0.00). In addition, several immune-related genes including DC marker gene, LRP1 (CD91; p < 0.00, FC = 1.3) and the neutrophil/GMP- specific gene, cathepsin G (CTSG; p < 0.01, FC = 0.5) showed a significant up-regulation in aplasia LAIP+ cells. The latter was previously identified as a leukemia-associated antigen and a potential target for immunotherapy (Alatrash et al., 2017). Strikingly, LAIP+ cells at the time of aplasia harbored a LSC gene expression pattern in a patient-specific manner. Lastly, cell type prediction using the annotated dataset (van Galen et al., 2019) assigned the phenotype of LAIP+ cells mainly to GMP-like and cDC-like cells in variable abundance across patients. In summary, our data showed high inter-patient heterogeneity and phenotypic differences, making general statements on chemo-resistance challenging. However, despite this heterogeneity, LAIP+ cells at the time of aplasia harbor remarkable proliferation and survival advantage (stem cell-like phenotype), specifically, due to the upregulation of SMARCA4, TRAP1, and LIN28B genes. This phenotype is accompanied by patient-specific enrichment of LSC signature and upregulation of immune-related genes. Our data open up the path to better understand chemo-resistance and to identify targets that need to be addressed to eradicate MRD and improve relapse-free survival. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Azacitidine Combined with Homoharringtonine, Idarubicin/Daunorubicin, Cytarabine for Previously Untreated Patients with Acute Myeloid Leukemia: A Single-Center, Phase 2 Study

Background Previous studies have reported that a combination of Homoharringtonine (HHT), a plant cytotoxic alkaloid derived from the trees of the genus Cephalotaxus, plus intensive chemotherapy (IC) improved the clinical responses of acute myeloid leukemia (AML). Azacitidine (Aza), a DNA hypomethylation agent, has been a "backbone" for novel combinational therapies. In this trial, we assessed the efficacy and safety of adding Aza to HIA/HDA regimen in previously untreated young or eligible for IC patients with AML. Methods This is a single-center, phase 2 trial (ClinicalTrials.gov: NCT04248595). Aza (75mg/m2/d on days1-5) administered in combination with HIA/HDA [HHT 2mg/m2/d on days 4-8, idarubicin 6mg/m2/d or daunorubicin 40 mg/m2/d on days 4-6, and cytarabine 100mg/m2/d on days 4-10] regimen for patients age<60 or eligible for IC (Fig.1A) The primary endpoint was the composite CR. Secondary endpoints were overall survival (OS), relapse-free survival (RFS), and adverse events (AEs). A leukemia targeted-exome-seq panel including 58 genes and leukemia 47 fusion gene panel were used for screening. Results Between Aug, 2019 to May, 2022, a total of 20 de novo AML patients were enrolled. The CR/CRi was achieved in 95.0% (19/20) (Fig. 1B). 94.7% (18/19) reached CR/CRi after the 1st cycle of induction therapy and the median time was 25.0d (IQR, 21.0 to 27.0). The median follow-up was 628 days (range, 107 to 905) and the median OS and RFS were not reached (Fig. 1C& 1D). The estimated 2-year OS and RFS were 87.4% (95%CI, 58.1% to 96.7%) (Fig. 1C) and 82.0% (95%CI, 53.1% to 94.0%), respectively (Fig. 1D). No survival difference was observed between the favorable and intermediate versus poor-risk groups in both OS and RFS (Fig. 1E& 1F). Meanwhile, the estimated 2-year OS and RFS possibility of the favorable and intermediate-risk group was 90.9% (95%CI, 50.8% to 98.7%) and 80.0% (95%CI, 20.4% to 96.9%), respectively (Fig. 1E& 1F). While the estimated 2-year OS and RFS possibility of the poor-risk group was 85.7% (95%CI, 33.4% to 97.9%), and 75.0% (95%CI, 12.8% to 96.1%), respectively (Fig. 1E& 1F). We detected 30 mutants in 17 of 58 leukemia driver genes at the timepoint of enrollment. The median mutation number/patient was 1.50 (range, 0-4), and 17 of 20 (85.0%) patients had more than one mutation (≥1) (Fig. 1G). The most frequently mutated genes were FLT3 (ITD/TKD) (20.0%), KIT (15.0%), NRAS (15.0%), and DNMT3A (15.0%) (Fig. 1G). More than half of the patients (11/20) harbored the mutated genes involved in signal transducers (FLT3, KRAS,NRAS, KIT, PTPN11, CSF3R; 11/20, 55.0%), and all these patients achieved CR/CRi (11/11, 100%) after induction therapy (Fig 1G). It is worth noticing that, we observed a high mutant's clearance with VAF (variant allele frequency) dramatical reduction (to <0.01%) (Fig. 1H) after the induction therapy, indicating that mutations involved in signal transducers were rapidly and significantly associated with high clinical response. We also identified 2 patients with MLL-AF9 and 4 patients with CBF-β-MYH11. Both patients with MLL-AF9 reached complete molecular remission (<0.1%) after induction. 3 of 4 patients with CBF-β -MYH11 reached complete molecular remission (<0.1%), and the expression level of the CBF-β -MYH11 fusion gene in 1 patient decreased over 1000-fold at the molecular level (512.16% to 0.28%) after 1 cycle of induction. The MRD negative was 84.2% (16/19) in patients who reached CR/CRi after the 1st cycle of induction therapy (Fig. 1I, 1J). Also, MRD were negative in all 3 patients with favorable risk, 77.8% with intermediate-risk, and 87.5% with poor-risk (Fig. 1K). Infection (grade 1-4) was the most common non-hematological AE (65.0%). Other AEs of grade 3 or higher included fatigue (5.0%), decreased appetite (5.0%), and hepatic dysfunction (5.0%). The most common grade 3-4 hematological AEs were neutropenia and thrombocytopenia. The median duration of neutropenia and thrombocytopenia were 12d (IQR, 11 to 15) and 14d (IQR, 12 to 16), respectively. No patients discontinued the induction treatment and no patient died within 8 weeks of the treatment. Conclusion This trial demonstrated that the adding of Aza to HIA/HDA regimen is an effective first-line therapy for previously untreated young or fit AML patients, with high efficacy and well tolerance. This study provides preliminary results for the clinical application of this novel combination therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Deep Molecular and In Silico Protein Analysis of p53 Alteration in Myelodysplastic Neoplasia and Acute Myeloid Leukemia.

Mutation of the TP53 gene is one of the major drivers of myelodysplastic neoplasias (MDS) and acute myeloid leukemia with myelodysplasia-related changes (AML-MR). TP53 mutations present in these hematopoietic malignancies form a distinct molecular genetic cluster with a worse prognosis than without the alteration. However, besides well-characterized hot-spot variants, a significant proportion of TP53 alterations are of uncertain clinical significance. To enlighten so far unknown aspects, bone-marrow samples from altogether 77 patients are analyzed retrospectively with the diagnosis of AML-MR (26 cases), MDS-IB (12 cases), and MDS-LB (39 cases) according to WHO 2022 guidelines. Next-generation sequencing results are correlated with histological, cytogenetic, and survival data. Twenty out of the 30 TP53 mutation types detected by NGS are not categorized in current public databases; thus, their clinical significance remained mysterious. Because of the interpretation difficulties and the absence of clinical correlations, pathogenicity is established based on in silico approaches. The 12 pathogenicity classification systems, as well as protein stability, protein-DNA, protein-protein interaction, and post-translational modification analyses are applied. We found statistically significant differences between AML/MDS groups considering p53 pathogenicity, protein structural changes, and overall survival. The largest number of abnormalities with the most severe consequences are found in AML-MR cases. These molecular and in silico protein data further support that MDS with increased-blast (MDS-IB) is an intermediate group between AML-MR and MDS with low-blast (MDS-LB) patients, which frequently progresses to AML and is therefore considered a pre-leukemic condition.

Hyperthermia promotes degradation of the acute promyelocytic leukemia driver oncoprotein ZBTB16/RARα.

The acute promyelocytic leukemia (APL) driver ZBTB16/RARα is generated by the t(11;17) (q23;q21) chromosomal translocation, which is resistant to combined treatment of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) or conventional chemotherapy, resulting in extremely low survival rates. In the current study, we investigated the effects of hyperthermia on the oncogenic fusion ZBTB16/RARα protein to explore a potential therapeutic approach for this variant APL. We showed that Z/R fusion protein expressed in HeLa cells was resistant to ATO, ATRA, and conventional chemotherapeutic agents. However, mild hyperthermia (42 °C) rapidly destabilized the ZBTB16/RARα fusion protein expressed in HeLa, 293T, and OCI-AML3 cells, followed by robust ubiquitination and proteasomal degradation. In contrast, hyperthermia did not affect the normal (i.e., unfused) ZBTB16 and RARα proteins, suggesting a specific thermal sensitivity of the ZBTB16/RARα fusion protein. Importantly, we found that the destabilization of ZBTB16/RARα was the initial step for oncogenic fusion protein degradation by hyperthermia, which could be blocked by deletion of nuclear receptor corepressor (NCoR) binding sites or knockdown of NCoRs. Furthermore, SIAH2 was identified as the E3 ligase participating in hyperthermia-induced ubiquitination of ZBTB16/RARα. In short, these results demonstrate that hyperthermia could effectively destabilize and subsequently degrade the ZBTB16/RARα fusion protein in an NCoR-dependent manner, suggesting a thermal-based therapeutic strategy that may improve the outcome in refractory ZBTB16/RARα-driven APL patients in the clinic.