<p>supplementary data Figure S1. Gene expression signatures define distinct molecular groups of T-ALL. Figure S2. Global flow chart of the patients Figure S3. Comparison of outcomes between LALA94 patients with central lab onco-genetic study performed (dotted line) and patients without (full line). Figure S4. Epigenetic histone marks. Figure S5. Kaplan-Meier graph for OS is shown for TAL1+ patients treated on LALA-94 vs. GRAALL-2003/2005 trials. Figure S6. TAL1 expression normalised to GAPDH by RTQ-PCR in 8 PDX treated with L-asparaginase (Rf. Figure 4H). Figure S7. (A) Correlation analysis between ASNS expression and ASNS promoter methylation ratio. (B) ASNS transcriptional expression normalized to GAPDH in T-ALL, in normal Bone Marrow (BM) (100%, 50%, 10% and 1%) and in normal Peripheral Blood Cells (PBL) (100%, 50%, 10% and 1. (C) comparison of ASNS transcriptional expression in non-sorted diagnostic sample, blast and non-blast sorted cells in two patients with a hypermethylated ASNS promoter (UPNT-573 and UPNT-498) and high ASNS expression (T3 tertile) (left) and, in primary samples and PDX (Primary Derived Xenograft) using a human specific Taqman system for two hypermethylated cases (UPNT-485 and UPNT-419) and two hypomethylated cases (UPNT-615 and UPNT-241) (right). (D) Correlation analysis between ASNS expression and ASNS promoter methylation ratio in purified blasts cells (PDX n=8 )and sorted primary samples (n=3). (E) Correlation analysis between ASNS expression (RNA-seq) and ASNS promoter methylation ratio in a series of 13 T-ALL cell lines (LOUCY, DND41, RPMI8402, HPB-ALL, ALL-SIL, TALL1, MOLT3, PEER, SUPT1, PF382, MOLT3, MOLT16, JURKAT). Table S1. Clinico-biological characteristics of patients included in the LALA94 trial according to their inclusion in the present study. Table S2. GRAALL03, 05 and LALA94 trials overview and drug administration schedule. Table S3. SiRNA sequences Table S4. Probes sequences for ASNS MS-MLPA analysis Table S5. DNA Global Methylation study Table S6. Characteristics of TLX1+, TLX3+ and TLX neg patients Table S7. Clinico-biological characteristics of T1 (lower ASNS methylation tertile) and T2+T3 (higher methylation tertiles) adult T-ALL. Table S8. Univariate and multivariate analysis for EFS and OS</p>
Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with a dismal prognosis in the case of primary refractory disease or relapse. The phenotypic and oncogenic landscapes of T-ALL are complex, hindering the design of efficient targeted therapy. Previously published results reported on the induction of apoptosis secondary to a chronic T cell receptor engagement with anti-CD3ε antibody (OKT3) via a signaling program related to developmental negative selection in sCD3+ leukemia models. Aims: We aim to evaluate the effect of a polyclonal IgG cocktail, thymoglobulin (ATG), on human T-ALL. We hypothesized that this treatment contains antibodies against different targets expressed at the cell surface of T-ALL (CD3 among others) and could potentially be an efficacious therapy for T-ALL. Methods: We tested the cytotoxicity of ATG in T-ALL cell lines and a series of human T-ALL ex vivo and in vivo (in curative settings) representing the different subtypes of all T-ALL in terms of phenotype and molecular landscape. We RNAsequenced sensitive and resistant cells exposed to ATG and compared results to control to identify enriched pathways after ATG exposure in both groups. Results: We screened a large series of 45 Patient-Derived-Xenograft (PDX) for their response to ATG. 27 T-ALL PDXs (60%) were responsive as early as 24h of exposure. This elevated response rate was obtained in most sCD3+ T-ALL (n=14/17), and strikingly, in a significant proportion of sCD3- T-ALL (n=13/28). We then tested in vivo 9 distinct human T-ALL PDX models, 2 of which expressed a surface CD3 (sCD3) and 7 were absent for sCD3. For all predicted ATG-responsive T-ALL, ATG treatment showed strong anti-leukemic effects, as demonstrated by the delayed dissemination of leukemic blasts in the blood and improved mice survival (Fig1A). We RNA-sequenced 7 T-ALL PDXs that were responsive ex vivo. Differential gene expression analysis in ATG vs control condition was performed and GSEA identified the TNFα signaling via NF-κB pathway as the only enriched pathway in these PDXs. No significant enrichment of this pathway was identified in resistant T-ALL (Fig1B). In line with this, activated caspase 8 was induced after ATG in ALL SIL and JURKAT responsive cell lines compared to the resistant cell line RPMI-8402. Similar results were also observed in 4 ATG-responsive PDXs. Moreover, after a shorter exposure to ATG, ALL-SIL and JURKAT (ATG-responsive) induced expression of pMLKLS358 protein, which plays a major role in TNFRSF-induced cell death through necroptosis unlike RPMI-8402. These results suggest the role of cell death induced by the TNF receptor superfamily leading to apoptosis (caspase 8 activation) or necroptosis (RIPK3 and MLKL activation) after ATG exposure (Fig1C). In parallel, we identified through RNAsequencing a cluster of genes constantly increased through ATG exposure (H1, H4, H24) and belonging to TNFα signaling via NF-κB pathway. The BIRC3 gene encoding for the cIAP2 protein was strongly induced after ATG in sensitive and resistant T-ALL PDX (n=3). We subsequently tested the effect of the combination of birinapant and ATG ex vivo in 22 PDXs. All six ATG-responsive benefited from the combination. Among the 16 ATG-resistant cases, 13 had a decreased cell viability but 3 remained resistant. Critically, all the PDXs resistant to Birinapant and ATG alone (n=11) obtained an additive (n=5) or synergic effect (n=6) with a significant decrease in cell viability (Fig1D). Summary/Conclusion: These results provide a strong rationale for the combination of SMAC mimetic with ATG immunotherapy inducing TNFα signaling in T-ALL.Keywords: T cell acute lymphoblastic leukemia
ALK-positive anaplastic large cell lymphoma (ALK+ ALCL) patients (pts) who have failed brentuximab vedotin (BV) have a poor prognosis with a median OS after BV failure of 2.9 months and 2-year OS of 27.1% (Chihara D, 2019). ALK-inhibitors have shown interesting results in relapsed ALK+ ALCL, but in these studies, most pts had not received prior BV, which does not correspond to current standards of treatment in adults. Furthermore, there are currently several ALK-inhibitors, but too few pts available to test them in this rare and difficult-to-treat population. It is therefore important to evaluate these ALK-inhibitors in preclinical studies, before selecting one for a clinical study. We carried out a preclinical study and then a real-life clinical study. We generated a patient-derived xenograft (PDX) model from a fresh lymph node biopsy of a 41-year-old man newly diagnosed with ALK+ ALCL. Our PDX closely mimicked the patient's primary tumor, as assessed by pathology, FISH, TCR gene rearrangement, WES and RNA-seq. We used this model to assess 8 ALK-inhibitors (alectinib, brigatinib, ceritinib, crizotinib, ensartinib, entrectinib, lorlatinib, gilteritinib). We selected and recommended brigatinib for clinical off-label use based on our preclinical results and the safety profile in pts with ALK-positive non-small cell lung cancer (NSCLC). Between Jan 2020 and Oct 2022, 15 French adults who have failed BV started brigatinib. At brigatinib initiation, the median age was 35 y (19–73; 2 pts > 60 y), 8/15 were male, the median number of prior treatment lines was 2 (1–8), 4/15 (27%) had received prior crizotinib, including 3 crizotinib-resistant (crizo-R) and 1 crizotinib-sensitive (crizo-S) who relapsed after discontinuation of the drug. 4 pts had previously undergone stem cell transplantation (3 autoSCT, 1 alloSCT). ALCL was refractory to the last treatment in 10/15 pts. 10/11 pts had detectable ALK transcript in blood by RT-PCR. Pts received brigatinib at a dose of 180 mg once daily (with a 7-day lead-in period at 90 mg), as recommended in ALK-positive NSCLC. The best ORR was 93% (14/15) with 73% (11/15) CR according to Lugano response criteria. 2 crizo-R and the crizo-S pts achieved CR, and 1 crizo-R pt reached PR. Time to achieve CR ranged from 8 to 325 days. 9 pts were monitored for ALK transcript in blood over time and kinetics correlated with response. 5 CR pts were bridged to alloSCT. There were 4 progressions/relapses after brigatinib initiation, all occurring within the first 6 months. After a median follow-up of 1.3 years, 1-year PFS and OS were 72% and 85%, respectively. There was no permanent discontinuation of brigatinib related to adverse event (AE), and 3 pts had dose reduction for moderate AE (1 dyspnea and 2 cramps), with complete resolution. Keywords: Aggressive T-cell non-Hodgkin lymphoma, Molecular Targeted Therapies Conflicts of interests pertinent to the abstract. D. Sibon Consultant or advisory role: Takeda, AbbVie, Janssen, Roche
<div>AbstractPurpose:<p>Biological explanation for discrepancies in patient-related response to chemotherapy depending on the underlying oncogenic events is a promising research area. TLX1- or TLX3-deregulated T-cell acute lymphoblastic leukemias (T-ALL; TLX1/3<sup>+</sup>) share an immature cortical phenotype and similar transcriptional signatures. However, their prognostic impacts differ, and inconsistent clinical outcome has been reported for TLX3. We therefore hypothesized that the overlapping transcriptional profiles of TLX1<sup>+</sup> and TLX3<sup>+</sup> T-ALLs would allow identification of candidate genes, which might determine their distinct clinical outcomes.</p>Experimental Design:<p>We compared TLX1<sup>+</sup> and TLX3<sup>+</sup> adult T-ALL outcome in the successive French national LALA-94 and GRAALL-2003/2005 multicentric trials and analyzed transcriptomic data to identify differentially expressed genes. Epigenetic regulation of asparagine synthetase (<i>ASNS</i>) and <i>in vitro</i> l-asparaginase sensitivity were evaluated for T-ALL cell lines and primary samples.</p>Results:<p>We show that TLX1<sup>+</sup> patients expressed low levels of <i>ASNS</i> when compared with TLX3<sup>+</sup> and TLX-negative patients, due to epigenetic silencing of <i>ASNS</i> by both DNA methylation and a decrease of active histone marks. Promoter methylation of the <i>ASNS</i> gene correlated with l-asparaginase sensitivity in both T-ALL cell lines and patient-derived xenografts. Finally, <i>ASNS</i> promoter methylation was an independent prognostic factor for both event-free survival [HR, 0.42; 95% confidence interval (CI), 0.24–0.71; <i>P</i> = 0.001] and overall survival (HR, 0.40; 95% CI, 0.23–0.70; <i>P</i> = 0.02) in 160 GRAALL-2003/2005 T-ALL patients and also in an independent series of 47 LL03-treated T lymphoblastic lymphomas (<i>P</i> = 0.012).</p>Conclusions:<p>We conclude that <i>ASNS</i> methylation status at diagnosis may allow individual adaptation of l-asparaginase dose.</p></div>
Cancer cells utilize the main de novo pathway and the alternative salvage pathway for deoxyribonucleotide biosynthesis to achieve adequate nucleotide pools. Deoxycytidine kinase is the rate-limiting enzyme of the salvage pathway and it has recently emerged as a target for anti-proliferative therapies for cancers where it is essential. Here, we present the development of a potent inhibitor applying an iterative multidisciplinary approach, which relies on computational design coupled with experimental evaluations. This strategy allows an acceleration of the hit-to-lead process by gradually implementing key chemical modifications to increase affinity and activity. Our lead compound, OR0642, is more than 1000 times more potent than its initial parent compound, masitinib, previously identified from a drug repositioning approach. OR0642 in combination with a physiological inhibitor of the de novo pathway doubled the survival rate in a human T-cell acute lymphoblastic leukemia patient-derived xenograft mouse model, demonstrating the proof-of-concept of this drug design strategy.
<p>supplementary data Figure S1. Gene expression signatures define distinct molecular groups of T-ALL. Figure S2. Global flow chart of the patients Figure S3. Comparison of outcomes between LALA94 patients with central lab onco-genetic study performed (dotted line) and patients without (full line). Figure S4. Epigenetic histone marks. Figure S5. Kaplan-Meier graph for OS is shown for TAL1+ patients treated on LALA-94 vs. GRAALL-2003/2005 trials. Figure S6. TAL1 expression normalised to GAPDH by RTQ-PCR in 8 PDX treated with L-asparaginase (Rf. Figure 4H). Figure S7. (A) Correlation analysis between ASNS expression and ASNS promoter methylation ratio. (B) ASNS transcriptional expression normalized to GAPDH in T-ALL, in normal Bone Marrow (BM) (100%, 50%, 10% and 1%) and in normal Peripheral Blood Cells (PBL) (100%, 50%, 10% and 1. (C) comparison of ASNS transcriptional expression in non-sorted diagnostic sample, blast and non-blast sorted cells in two patients with a hypermethylated ASNS promoter (UPNT-573 and UPNT-498) and high ASNS expression (T3 tertile) (left) and, in primary samples and PDX (Primary Derived Xenograft) using a human specific Taqman system for two hypermethylated cases (UPNT-485 and UPNT-419) and two hypomethylated cases (UPNT-615 and UPNT-241) (right). (D) Correlation analysis between ASNS expression and ASNS promoter methylation ratio in purified blasts cells (PDX n=8 )and sorted primary samples (n=3). (E) Correlation analysis between ASNS expression (RNA-seq) and ASNS promoter methylation ratio in a series of 13 T-ALL cell lines (LOUCY, DND41, RPMI8402, HPB-ALL, ALL-SIL, TALL1, MOLT3, PEER, SUPT1, PF382, MOLT3, MOLT16, JURKAT). Table S1. Clinico-biological characteristics of patients included in the LALA94 trial according to their inclusion in the present study. Table S2. GRAALL03, 05 and LALA94 trials overview and drug administration schedule. Table S3. SiRNA sequences Table S4. Probes sequences for ASNS MS-MLPA analysis Table S5. DNA Global Methylation study Table S6. Characteristics of TLX1+, TLX3+ and TLX neg patients Table S7. Clinico-biological characteristics of T1 (lower ASNS methylation tertile) and T2+T3 (higher methylation tertiles) adult T-ALL. Table S8. Univariate and multivariate analysis for EFS and OS</p>
Background: Normal T‐cell development is supported by cytokine signaling with IL7 as key player. This is highlighted by the absence of T‐cells in gamma‐chain SCID‐patients. T‐ALL represents a heterogeneous disease characterized by expansion of immature T‐cells blocked in their differentiation. Despite therapeutic improvements, it remains associated with a poor prognosis mainly due to relapses. This results in a medical need for new therapeutic approaches. Several studies reported the deregulation of IL7R signaling notably via genomic alterations accounting for ∼30% of T‐ALL. In keeping with this, preclinical models supported the possibility to target the IL7R‐pathway (IL7Rp) in IL7Rp‐mutated T‐ALL using pharmacological JAK inhibitors such as ruxolitinib. Moreover, ex vivo culture of primary T‐ALL samples requires addition of IL7 cytokine in culture medium to support leukemic cell survival. Altogether, this data supports a strong dependency of T‐ALL on IL7R signalling. However, the question of which T‐ALL patients may benefit from JAK inhibitors remains unclear. Aims: We aimed to investigate surface IL7R (sIL7R) expression and mutational status of IL7Rp in T‐ALL, and to identify a readout for patient JAK inhibitor sensitivity. Methods: 159 T‐ALL samples were prospectively analyzed by FACS for IL7R expression. Genetic data of IL7R pathway genes (IL7R, JAK1/2, STAT5) were assessed on 87 adult T‐ALL enrolled in the ongoing GRAALL‐2014 trial by targeted‐sequencing. 21 patient derived xenograft (PDX) T‐ALL were generated for ex vivo studies including i) phosphoprotein flow analysis of pSTAT5 upon IL7 stimulation and JAK inhibition, ii) apoptosis assay by Annexin V‐PI staining, and iii) proliferation assay using Celltrace. Results: sIL7R was expressed at early stages of normal T‐cell development, downregulated during the cortical stage, and up‐regulated at the mature stage. In T‐ALL, sIL7R was expressed in 84/159 cases (53%). We found IL7Rp mutations in 29% of cases and all IL7R mut cases expressed sIL7R defining 3 classes of T‐ALL depending on sIL7R expression and mutational status of IL7Rp: IL7R ‐/WT , IL7R +/WT , IL7R +/mut . IL7R + T‐ALLs were mainly of immature non‐ETP, cortical and TCRgd phenotype. These data support frequent and abnormal expression of sIL7R in T‐ALL as regard to normal expression pattern in thymocytes. sIL7R expression was always associated with functional signaling of the receptor: pSTAT5 was activated upon IL7 exposure and abrogated by the addition of ruxolitinib in both normal thymocytes and PDX T‐ALL. Constitutive STAT5 phosphorylation was observed in IL7R +/mut but not in IL7R +/WT PDX T‐ALL. However, pSTAT5 activation reached similar levels in both IL7R +/mut and IL7R +/WT PDX T‐ALL upon IL7 exposure regardless of their mutational status, which was completely reversed by ruxolitinib addition. Ex vivo treatment of PDX T‐ALL with increasing doses of ruxolitinib induced apoptosis and cytostatic effect in both IL7R +/WT and IL7R +/mut PDX T‐ALL. Summary/Conclusion: IL7Rp genes are mutated in 29% in T‐ALL and sIL7R is expressed in 53% of cases. Ruxolitinib induces apoptosis and impairs cell proliferation in sIL7R + T‐ALL irrespective of IL7Rp mutational status. Our data shows that sensitivity to JAK inhibitors is determined by the expression of IL7R regardless of the IL7Rp genomic status. IL7R expression is a readout for JAK inhibitors sensitivity, and provides a fast and accurate companion theranostic tool using flow‐cytometry.
