Paietta, Elisabeth

Background: Inotuzumab Ozogamicin (InO) is an antibody-cytotoxin conjugate with high activity in B-cell acute lymphoblastic leukemia (B-ALL). InO targets the B-cell surface protein CD22, which is expressed in most B-ALL cases, albeit with great variability in expression levels. Emerging evidence suggest that this variation is associated with the stage of leukemia differentiation arrest [PMID: 21348573]. However, the exact relationship between CD22 expression and leukemia sensitivity to InO, particularly in the context of leukemia developmental state, remains unclear. Furthermore, our understanding of the molecular mechanisms regulating CD22 transcription in B-ALL is still incomplete. Methods: To comprehensively investigate the genomic basis of variability in leukemia CD22 expression and thus B-ALL sensitivity to InO, we performed a multi-omic characterization of 196 primary human B-ALL samples, using whole transcriptome sequencing (RNAseq), assay for transposase-accessible chromatin with sequencing (ATAC-seq), coupled with ex vivo profiling of InO sensitivity. Additionally, genomic editing studies were performed in Nalm6 and REH B-ALL cell lines, and patient derived xenograft (PDX) models were used for in vitro and in vivo validations. Results: Applying digital cytometry to leukemia bulk RNAseq data to infer the B-ALL developmental state, we found that blast cells with early Pre-pro-B-like signature had low levels of CD22 expression and were resistant to InO. As B-cell differentiation is controlled by stage-specific transcription factors (TFs), we performed a targeted CRISPR library screen of 1,639 TF genes and identified EBF1 as a key activator of CD22 expression (FDR=7.1x10-4). In primary B-ALL samples, EBF1 expression was significantly correlated with CD22 expression (R=0.69, P<0.0001) and leukemia response to InO (measured as LC50, R=-0.42, P<0.0001). In addition, TF footprint analysis of leukemia ATAC-seq data identified EBF1 as the most differentially activated TF comparing InO-sensitive and -resistant B-ALL cases (P=8x10-174). Blocking the EBF1 binding sites within the CD22 promoter by CRISPR interference resulted in a 50-fold and 49-fold decrease in cell surface CD22 expression, and a 22-fold and 25.7-fold increase in InO LC50 for Nalm6 and REH, respectively, confirming the direct impact of EBF1 on CD22 transcription and InO sensitivity. Because BCR::ABL1-positive ALL showed the greatest variability in InO sensitivity among all B-ALL subtypes, we further investigate the role of the EBF1/CD22 axis on InO sensitivity within this subtype. Unsupervised clustering of gene expression profiles revealed two distinct groups, namely BCR::ABL1 ALL with multilineage (BCR::ABL1-M) or lymphoid only (BCR::ABL1-L) involvement [PMID: 38153913]. Compared to BCR::ABL1-L, BCR::ABL1-M displayed markedly reduced CD22 expression (P=8.3×10-11) and resistance to InO (P=1.9×10-4), likely due to the lower expression and frequent somatic alteration of EBF1. Finally, we established PDX models for representative cases of BCR::ABL1-M and BCR::ABL1-L. Strikingly, InO treatment did not affect leukemia progression in the BCR::ABL1-M PDX model, whereas the BCR::ABL1-L PDX model achieved complete remission and has remained leukemia free to date. Conclusion: Collectively, these data demonstrate the direct impact of EBF1 on CD22 expression during B-cell development, and explain the heterogeneity of InO sensitivity, even within the same molecular B-ALL subtype.

AbstractIntroductionRelapsed or refractory (r/r) acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LL) remains a therapeutic challenge. Preclinical data in both B‐ and T‐ALL suggests synergy of venetoclax (VEN) with vincristine (VCR). We designed a phase I/II trial (EA9152) of the combination of L‐VCR and VEN for patients with r/r B‐or T‐cell ALL or LL. Here, we report the safety and efficacy outcomes of the phase I portion of this trial (NCT03504644).MethodsIn a 3+3 dose escalation design, r/r ALL subjects were given single‐agent VEN doses reaching 400, 600, or 800 mg for the three respective dose levels. Weekly L‐VCR at 2.25 mg/m2 IV was started on D15 of cycle 1. The primary phase I objective was to determine the maximum tolerated dose (MTD) of the combination.ResultsAmong the 18 patients in phase I, grade ≥ 3 treatment‐related adverse events were reported in 89% of treated patients. Two patients (two of three) at dose level 3 experienced dose‐limiting toxicities. Therefore, the MTD of the combination was determined to be dose level 2 (VEN 600 mg). Twenty‐two percent of evaluable patients (N = 4) achieved a complete response, with two of them showing no evidence of measurable residual disease (MRD).ConclusionThe combination of VEN and L‐VCR was found to be safe for patients with r/r ALL and encouraging preliminary efficacy, including MRD negative responses. With the removal of L‐VCR from the US market, the phase 2 portion of this trial is actively enrolling with vincristine sulfate.

While the cure rate of pediatric acute lymphoblastic leukemia (ALL) now exceeds ~90% with contemporary combination chemotherapy, the prognosis for adults with ALL remains significantly inferior with long-term overall survival ranging from 50% to 70%. Recent studies have uncovered marked differences in ALL genomics between children and adults, with some high-risk subtypes becoming more prevalent with age. However, the underlying biology of age-related disparities in ALL is not fully understood, especially with regard to differences in leukemia sensitivity to chemotherapy. To address this knowledge gap, we performed ex vivo drug sensitivity profiling (i.e., pharmacotyping) of 21 anti-leukemia agents on primary B-ALL diagnostic samples from 767 pediatric (age, 0-18 years) and 309 adult (19-84 years) patients. Drug sensitivity was measured as LC 50: the concentration of drug required to kill 50% of the leukemia cells (PMID: 36604538). A total of 7,975 unique LC 50 values were experimentally determined. RNA-seq was used for subtype classification and gene expression analysis. Among 21 drugs, seven showed significant differences in overall LC 50 between children and adults ( P<0.05 after Bonferroni correction): children displayed higher sensitivity to asparaginase, prednisolone, mercaptopurine, daunorubicin, and inotuzumab, while adults showed higher sensitivity to dasatinib and nelarabine. In multivariate models adjusting for 23 ALL molecular subtypes, only mercaptopurine remained significantly associated with age ( P=1.5×10 -5), suggesting that age-related differences in drug sensitivity can be primarily attributed to the variation in ALL subtypes between children and adults. For mercaptopurine, within KMT2A, CRLF2, and DUX4 subtypes, pediatric samples consistently showed a lower LC 50 than adults carrying the same genomic abnormality ( P=0.032, 0.0045, and 0.02, respectively). To explore intra-subtype heterogeneity, we performed unsupervised clustering using gene expression data for each of these three subtypes. Remarkably, within each of these subtypes, we identified two clusters with distinct transcriptomic profiles that were also largely segregated by age group, i.e., an adult-dominated cluster (C-a) and a pediatric-dominated cluster (C-p). In the KMT2A subtype, cases in C-a exhibited an over-representation of the KMT2A:: AFF1 fusion, and resistance to mercaptopurine ( P=0.029), prednisolone ( P=0.0039), vincristine ( P=0.046) and cytarabine ( P=0.0037). Within CRLF2 ALL, cases in C-a were associated with the presence of BCR:: ABL1-like signature and IGH:: CRLF2 rearrangements, and were more resistant to mercaptopurine ( P=0.0073) and prednisolone ( P=0.00036) compared to those in C-p. For DUX4 ALL, C-a was characterized by an under-representation of ERG deletions and resistance to mercaptopurine ( P=0.0056) and prednisolone ( P=0.0031), compared to C-p within DUX4. To explore the clinical relevance of this heterogeneity, we analyzed the in vivo treatment response of KMT2A (N=35), CRLF2 (N=59) and DUX4 (N=118) B-ALL enrolled in six frontline ALL trials. Compared to cases in C-p (usually drug-sensitive), those in C-a (usually drug-resistant) consistently had significantly poorer initial treatment responses as measured by persistent end-of-induction minimal residual disease (≥0.01%) in KMT2A (58% vs 9%; P=0.0063), CRLF2 (74% vs 41%; P=0.030), and DUX4 (66% vs 40%; P=0.0058) ALL. In conclusion, these studies have revealed important new insights into the pharmacogenomic basis of age-related differences in B-ALL treatment response. These results indicate that both inter- and intra-subtype heterogeneity contribute to inferior prognosis in adults with ALL, but also point to therapeutic opportunities to improve their outcomes.

Introduction: ECOG ACRIN E1910 is a randomized phase III trial that showed that adults with newly diagnosed BCR::ABL1 negative acute lymphoblastic leukemia (ALL) who become MRD negative (<0.01%) after induction chemo who receive blinatumomab with conventional chemotherapy (chemo) have improved survival compared with those who received chemo only (Litzow et al, Blood (2022) 140: Supplement 2, LBA-1). However, not all pts were able to receive all four planned cycles of blinatumomab in consolidation. In this report we assessed outcomes of pts in the blinatumomab arm of the trial who received all 4 cycles of blinatumomab compared to those who received 1-2 cycles. Methods: Patient between the ages of 30 and 70 with newly diagnosed BCR::ABL1 negative B-lineage ALL were enrolled and initially received 2.5 months of combination induction chemo utilizing a BFM-like regimen adapted from the E2993/UKALLXII clinical trial, with pegaspargase (after induction only for patients >=55 years of age) and addition of rituximab for CD20 positive patients. After remission induction (step 1) pts in morphologic complete remission (CR/CRi) received an intensification course of high dose methotrexate with pegaspargase for CNS prophylaxis (step 2). At the conclusion of step 2, remission and MRD status were determined centrally by 6-color flow cytometry with MRD negativity defined as <0.01%. In the primary analysis subset, MRD negative patients were randomized to receive an additional 4cycles of consolidation chemo or 2 cycles of blinatumomab at 28 mcg/day (flat dose) for 28 days each cycle followed by 3 cycles of consolidation chemo, another 4-week cycle of blinatumomab (3rd cycle of blinatumomab) followed by an additional cycle of chemo and then a 4th cycle of blinatumomab (step 3). Following completion of consolidation chemo +/- blinatumomab, pts were given 2.5 years of POMP maintenance therapy timed from the start of the intensification cycle (step 4). Estimates of OS were calculated using the Kaplan-Meier method. Comparison of OS between treatment arms were conducted using the stratified log-rank test and Cox proportional-hazards model with age, CD20 status, rituximab use, and whether pts intend to receive HSCT or not as stratification factors. As the number of cycles needed to optimize outcome has not been studied, an unplanned landmark analysis was used to compare OS of pts in the blinatumomab arm who received all 4 cycles o those who received 1-2 cycles. Time 0 was chosen as 9 months post step 3 randomization (the time that patients were supposed to complete 4 cycles of blina). Four pts who received blinatumomab but died within 9 months post step 3 randomization were therefore excluded from this analysis. Results: The study was activated on December 17, 2013 and closed to enrollment on October 15, 2019. 772 pts were screened for the trial and 488 were enrolled on step 1 induction therapy. The median age of the pts was 51 years (range 30-70). 224 MRD negative pts were randomized, 112 pts to each arm. In the blinatumomab arm 12 pts received 1 cycle (11%), 32 pts received 2 cycles (29%), 4 pts received 3 cycles (4%) and 63 pts received 4 cycles (57%). The OS of pts who received 1-2 cycles of blinatumomab was compared to control (Figure 1) The difference was not significant (hazard ratio 0.62, 95% CI 0.28 to 1.34, p=0.22). Fig 2 compares the survival of those who received 1-2 cycles to those who received 4 cycles ( (HR: 0.39, 95% CI 0.12 to 1.16, p=0.076). Conclusion: The addition of blinatumomab to consolidation chemotherapy resulted in a significantly better overall survival compared to chemotherapy alone in pts with newly diagnosed B-lineage ALL who were MRD negative after intensification. This represents a new standard of care for pts aged 30-70 years with BCR::ABL1 negative ALL. The optimal dose and number of cycles is however unknown. In this unplanned subgroup analysis we demonstrate that a survival benefit can only be confirmed in patients who receive the intended 4 cycles of blinatumomab during consolidation. Further studies will be needed to determine if fewer cycles can provide a similar benefit.

AbstractBackgroundObesity (body mass index [BMI] ≥30 kg/m2) is an important epidemiological risk factor for developing acute myeloid leukemia (AML). Therefore, the authors studied the association of obesity with clinical and genetic phenotype and its impact on outcome in adults with AML.MethodsThe authors analyzed BMI in 1088 adults who were receiving intensive remission induction and consolidation therapy in two prospective, randomized therapeutic clinical trials of the Eastern Cooperative Oncology Group‐American College of Radiology Imaging Network: E1900 (ClinicalTrials.gov identifier NCT00049517; patients younger than 60 years) and E3999 (ClinicalTrials.gov identifier NCT00046930; patients aged 60 years or older).ResultsObesity was prevalent at diagnosis (33%) and, compared with nonobesity, was associated with intermediate‐risk cytogenetics group (p = .008), poorer performance status (p = .01), and a trend toward older age (p = .06). Obesity was not associated with somatic mutations among a selected 18‐gene panel that was tested in a subset of younger patients. Obesity was not associated with clinical outcome (including complete remission, early death, or overall survival), and the authors did not identify any patient subgroup that had inferior outcomes based on BMI. Obese patients were significantly more likely to receive <90% of the intended daunorubicin dose despite protocol specification, particularly in the E1900 high‐dose (90 mg/m2) daunorubicin arm (p = .002); however, this did not correlate with inferior overall survival on multivariate analysis (hazard ratio, 1.39; 95% confidence interval, 0.90–2.13; p = .14).ConclusionsObesity is associated with unique clinical and disease‐related phenotypic features in AML and may influence physician treatment decisions regarding daunorubicin dosing. However, the current study demonstrates that obesity is not a factor in survival, and strict adherence to body surface area‐based dosing is not necessary because dose adjustments do not affect outcomes.

Leukemia cells accumulate DNA damage but altered DNA repair mechanisms protect them from apoptosis. We showed here that formaldehyde generated by serine/one-carbon cycle metabolism contributed to accumulation of toxic DNA-protein crosslinks (DPCs) in leukemia cells, especially in driver clones harboring oncogenic tyrosine kinases [OTKs: FLT3(ITD), JAK2(V617F), BCR/ABL1]. To counteract this effect, OTKs enhanced the expression of DNA polymerase theta (POLq) by ERK1/2 serine/threonine kinase-dependent inhibition of c-CBL E3 ligase-mediated ubiquitination of POLq and its proteasomal degradation. Overexpression of POLq in OTK-positive cells resulted in efficient repair of DPC-containing DNA double-strand breaks (DSBs) by POLq-mediated end-joining (TMEJ). Transforming activity of OTKs and other leukemia-inducing oncogenes, especially of those causing inhibition of BRCA1/2 -mediated homologous recombination (HR) with and without concomitant inhibition of DNA-PK -dependent non-homologous end-joining (D-NHEJ), was abrogated in Polq-/- murine bone marrow cells. Genetic and pharmacological targeting of POLq polymerase and helicase activities revealed that both activities are promising targets in leukemia cells. Moreover, OTK inhibitor or DPC-inducing drug etoposide enhanced anti-leukemia effect of POLq inhibitor (POLqi) in vitro and in vivo. In conclusion, we demonstrated that POLq plays an essential role in protecting leukemia cells from metabolically induced toxic DNA lesions triggered by formaldehyde and that it can be targeted to achieve therapeutic effect.

Blinatumomab is an efficacious immunotherapeutic agent in B cell acute lymphoblastic leukemia (B-ALL). However, the pharmacogenomic basis of leukemia response to blinatumomab is unclear. Using genome-wide CRISPR, we comprehensively identified leukemia intrinsic factors of blinatumomab sensitivity, i.e., the loss of CD58 as a top driver for resistance, in addition to CD19 . Screening 1639 transcription factor genes, we then identified PAX5 as the key activator of CD58. ALL with the PAX5 P80R mutation also expressed the lowest level of CD58 among 20 ALL molecular subtypes in 1988 patients. Genome editing confirmed the effects of this mutation on CD58 expression and blinatumomab sensitivity in B-ALL, with validation in patient leukemic blasts. We described a PAX5-driven enhancer at the CD58 locus, which was disrupted by PAX5 P80R, and the loss of CD58 abolished blinatumomab-induced T cell activation with global changes in transcriptomic/epigenomic program. In conclusion, we identified previously unidentified genetic mechanisms of blinatumomab resistance in B-ALL, suggesting strategies for genomics-guided treatment individualization.