Early T-cell Precursor Acute Lymphoblastic Leukemia Research Articles

Clinical features of immature leukemias in children.

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL), mixed phenotypic acute leukemia (MPAL), and acute myeloid leukemia with minimal differentiation (AML-M0) all originate from immature hematopoietic progenitor cells and have a poor prognosis. We investigated the clinical characteristics of these immature leukemias in 17 children (ETP-ALL: 8, MPAL: 5, AML-M0: 4) at seven institutions. Clinical and laboratory findings were comparable across disease types. Eleven and six patients received ALL- and AML-oriented induction chemotherapy, with six and four achieving complete remission (CR), respectively. Five additional patients achieved CR after salvage with the other type of chemotherapy. Eight patients received hematopoietic cell transplantation (HCT) in first CR, and six survived without relapse. However, six of seven patients who did not receive HCT during first CR relapsed; all underwent HCT later, and only three survived. The 5-year event-free survival (EFS) and overall survival (OS) rate were 37% and 69%, respectively. Patients who achieved CR after induction chemotherapy and received HCT in first CR had favorable EFS and OS. Notably, all patients who received HCT in first CR survived 5years after diagnosis. Appropriate induction chemotherapy and HCT in first CR could improve the outcome of immature leukemias.

Newly Dignosed Early T-Cell Precursor Acute Lymphoblastic Leukemia Successfully Treated with Venetoclax Combined with DA and IA Regime

Abstract Background: Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is a distinctive and extremely aggressive subtype of T-ALL. It is characterized by the aberrant expression of myeloid/stem cell antigens and the lack of Tlymphoid antigens including CD1a, CD4, CD5, and CD8. Currently, standard combination chemotherapy is still the mainstay of ETP-ALL treatment. However, the optimal management remains to be determined. ETP-ALL has low response rates to conventional chemotherapy and high relapse rates with a grim prognosis. Although some ETPALL patients achieved morphologic complete remission (CR), a more signifcant proportion maintained MRD positive. Under the traditional induction and intensifcation treatment strategy, the ETP-ALL group had worse long-term outcomes than the non-ETP-ALL group [1]. A recent preclinical study suggested that inhibition of B-cell lymphoma/leukemia-2 (BCL-2) might be a potential treatment option for patients with ETP-ALL [2]. Here, we present two cases of ETP-ALL clinical reactivity to venetoclax in combination with the IA regimen, providing a better bridge to allogeneic hematopoietic stem cell transplantation (allo-HSCT). Case 1 A 47-year-old female was diagnosed with ETPALL in October 2021. Flow cytometry analysis showed CD7,CD117,CD34,CD13,CD2,CD200,cyCD3 positive,suggestive of ETP ALL. Cytogenetic analysis was normal and molecular panel identified aberrations in TET, EP300 and EZH2. She then recived venetoclax plus DA regime（venetoclax, 100 mg d1, 200 mg d2, 400 mg qd d3-14；daunorubicin 40mg d1-3；cytarabine 100mg d1-5). A bone marrow biopsy after the 1st cycle revealed CR with minimum residual disease (MRD) 1.91% by flow cytometry. She then received 2nd cycle of venetoclax plus CAG treatment and got MRD negative. She received 2cycles of the same combination and remained in MRD-negative CR prior to proceeding with a reduced intensity haploidentical allo-transplantation. She is currently 1 years and 4 months post-transplant and in remission. Case 2 A 53-year-old man was diagnosed with ETP-ALL following bone marrow aspiration in August 2022. He presented with Leukopenia and anemia (WBC 2.53× 109 /L, HB 73 g/L, PLT 148 × 109 /L). Bone marrow biopsy revealed a hypercellular bone marrow with 80% lymphoid blasts, negative for MPO staining. The flow cytometry showed the immunophenotype of the lymphoid blasts (CD7+CD34+CD33+CD13+CD200+cyCD3+nTdT+), which were consistent with the immunophenotype of the ETP-leukemic cells. Cytogenetics revealed der(4)(q35).The molecular panel identified aberrations in CSF3R,DNMT3A,CCND3,CSF3R and WT1. Therefore, ETP-ALL was diagnosed. Initially, he received the first cycles of venetoclax combined with IA regimen (venetoclax, 100 mg d1, 200 mg d2, 400 mg qd d3-14;idarubicin 10mg d1-3,cytarabine 100mg d1-5), and CR was achieved this time. Then, he received a 3rd CAG therapy. Subsequently, he underwent hematopoietic stem cell transplantation (HSCT) successfully in December, and CR was still maintained at now 7 months after HSCT. Conclusion Combination therapy of venetoclax with IA regimen is an effective regimen for treating patients with ETP ALL.

Jmjd3 Drives Immature T-Cell Lymphoblastic Leukemia

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that accounts for 15% of pediatric and 25% of adult ALL cases. Despite an improved survival rate due to intensified chemotherapy and bone marrow transplantation, still 20% of T-ALL patients are primary refractory to the standard treatment or relapse, and have a poor clinical outcome. T-ALL patients can be classified into molecular genetic subgroups based on the expression or genomic alterations of specific oncogenic transcription factors. One such subgroup the early T-cell precursor ALL (ETP-ALL) is a heterogeneous subgroup of immature leukemias that often display high expression of LYL1 and LMO2. It's important to improve our knowledge of T-ALL biology and developing novel targeted anti-TALL therapies. We previously showed that the H3K27 demethylase Jmjd3 plays an important role in T-ALL and is a good candidate for the treatment of T-cell leukemia. Aim: Identify the role of Jmjd3 in the initiation of immature T-ALL. Methods: We generated a conditional Rosa26- Jmjd3 overexpression mouse model (Figure 1A), and crossed it with a hematopoietic-specific Vav-icre mice, to obtain Jmjd3 Vav mice. To investigate the role of Jmjd3 in tumor initiation, we monitored survival of a Jmjd3 Vav cohort. Arising tumors were characterized by immunohistochemistry, flow cytometry, and were transplanted into secondary immunodeficient recipients. To characterize the pre-leukemic compartment, we performed single cell feature barcoding and RNA sequencing. Also bulk RNAseq and proteomics were performed on primary and transplanted Jmjd3-driven tumors. To investigate the role of Jmjd3 in immature T-cell biology in human and mice, we analyzed thymi from 8-weeks-old control and Jmjd3 Vavmice, and performed OP9-DL4 stromal co-cultures with transduced CD34+ human cord blood cells that overexpressed JMJD3, respectively. We used ETP-ALL patient-derived xenografts (PDX) models to validate markers identified in single cell analysis. Results: We found that hematopoietic Jmjd3 overexpression resulted in hematological malignancies with 38% penetrance (n= 8/21) and a median survival of 63 weeks (Figure 1B). Immunohistochemistry confirmed overexpression of Jmjd3, decreased levels of H3K27me3, and increased levels of H3K27ac in Jmjd3 Vav tumors. Immunophenotypic analysis revealed immature Thy1.1 +CD4 -CD8 - cells in all obtained Jmjd3 Vav tumors (Figure 1C), indicative of an immature the early T-cell precursor (ETP)-like ALL. These immature T-ALLs gave rise to primary (7 weeks) and secondary (3 weeks) transplants upon their transplantation in immunocompromised mice. Bulk RNAseq confirmed that the transcriptional feature of the primary tumor were maintained upon transplantation. The role of Jmjd3 in stimulating immature thymocyte development was confirmed in thymi from 8-weeks-old Jmjd3 Vavmice and could be validated as well in human T-cell development using stromal OP9-DL4 co-cultures. Finally, we used single cell sequencing technologies to characterize both transcriptional features and cell-surface markers within the tumor fraction (Figure 1D). We confirmed that the tumor cluster was ETP-like with absence of CD1, CD5 and mature T-cell markers, and expression of CD44, Lyl1, and myeloid markers. Furthermore, we could identify cell-surface in immature Jmjd3-driven tumors that were also present in human T-cell stromal culture and ETP-ALL PDXs. Conclusion: We identified Jmjd3 as a driver of immature thymocyte populations that give rise to ETP-ALL.

Frontline Venetoclax Therapy for 18-Year-Old Male with Ataxia Telangiectasia and Early T-Cell Precursor Acute Lymphoblastic Leukemia

Ataxia Telangiectasia (AT) is an autosomal recessive disorder which is associated with progressive neurodegeneration, immunodeficiency, and predisposition to cancer. Specifically, around 40% of patients with AT will develop malignancy during their lifetime, most commonly lymphoid malignancies. Patients with AT have increased toxicity to chemotherapy and radiation, which necessitates modified and individualized therapy approaches. Early T-cell precursor ALL (ETP-ALL) is considered high risk and venetoclax has emerged as potential therapy combined with other chemotherapy to help achieve remission but there have been no reports of venetoclax use in patients with AT. Here, we present a case of an 18-year-old male with Ataxia Telangiectasia diagnosed with ETP-ALL, who achieved remission after therapy was augmented with venetoclax during consolidation phase of chemotherapy. The patient initially presented with lower limb swelling, fatigue and nose bleeds and found to have a white cell count (WBC) of 107 K/uL with 72% peripheral blasts. He was treated with steroid monotherapy for 38 days prior to presentation to our institution. On arrival to our institution he had a WBC count of 42 K/uL with 75 % blasts in peripheral blood. His bone marrow evaluation had 83% blasts. Flow cytometry was positive for CD3, CD5 (partial), CD7, CD38, CD45, CD54, CD56, CD 117 (partial), negative for MPO, TdT, HLA-DR. This confirmed the diagnosis of ETP-ALL. An 81 gene panel testing was positive for NOTCH 1, SRSF2, EZH2, CREBBP. No abnormal fusions or translocations were present, and cytogenetics did not reveal a clonal abnormality. Induction chemotherapy was given as per COG AALL1231 protocol with dose modifications including 50% decrease in vincristine and cyclophosphamide. His end of induction (EOI) bone marrow evaluation was positive for 32% MRD. Molecular diagnosis positive for NOTCH1, SRSF2 and EZH2. As it is not uncommon for patients with ETP-ALL to have persistent disease after induction chemotherapy cycle, we proceeded with consolidation per AALL0434 protocol with Nelarabine. Unfortunately, even with dose reduced vincristine he developed vocal cord paralysis, lower extremity neuropathy and urinary retention, hence vincristine was omitted. Bone marrow evaluation was repeated mid consolidation and was still positive for 20% MRD. Due to significant modifications in therapy as well as persistent disease midway through consolidation, venetoclax was added to augment therapy during the consolidation phase. He was given 150 mg/m2 for 7 days directly followed by nelarabine. End of consolidation marrow was negative for MRD, and molecular testing (81 gene panel) was also negative. He tolerated venetoclax well without any significant toxicities and remains in remission 4 months post therapy with venetoclax. This report indicates that venetoclax can be used safely in patients with AT. This patient showed persistent MRD positive mid-way through consolidation with negativity after augmentation with venetoclax. Patients with AT have increased risk of toxicity from chemotherapy agents which requires dose modifications in their chemotherapy regimen. Venetoclax augmentation may be a viable approach for patients with AT or DNA repair disorders.

Inflammatory Signatures Define a New High-Risk T-Lineage ALL Subtype

T-lineage acute lymphoblastic leukemia (ALL) is an aggressive cancer comprising of diverse subtypes that are challenging to stratify using conventional immunophenotyping and have historically exhibited poor treatment outcomes in response to cytotoxic chemotherapy ( Nguyen, K et al. Leukemia 2008). Early T-cell Precursor ALL (ETP-ALL) represent a distinct subtype that displays a developmental block at the earliest stages of T-cell commitment, accompanied by the aberrant expression of myeloid and stem cell markers ( Coustan-Smith, E. et al. Lancet Oncol. 2009). The classification can be further complicated by T/Myeloid Mixed phenotype acute leukemia (T/My-MPAL), a rare and aggressive malignancy characterized by blasts with both T-cell lymphoid and myeloid markers ( George, B. S. et al. Biomedicines 2022). Accurate clinical diagnosis of T-lineage ALL is hindered by phenotypic variations among its subsets. Nevertheless, the precise diagnosis holds critical importance as drug sensitivity in preclinical models of T-lineage ALL is closely linked to the differentiation state, such as the sensitivity of ETP-ALL to the BCL-2 inhibitor venetoclax ( Chonghaile, T. N. et al. Nat Cancer 2021). This highlights the critical need to identify consistent phenotypic traits associated with unique therapeutic vulnerabilities to effectively tailor therapy to individual patients. To gain insights into subset-specific therapeutic vulnerabilities, we performed an integrative multiomic analysis of bone marrow (BM) samples (n=21) from newly diagnosed T-lineage ALL patients, including T-cell ALL (T-ALL), ETP-ALL, and T/My-MPAL. Leveraging cellular indexing of transcriptomes and epitopes in conjunction with T-cell receptor sequencing, we identified a distinct subset of patients spanning all three subtypes characterized by the high frequency of leukemic cells that most closely resemble healthy hematopoietic stem and progenitor cells (HSPC) by Symphony ( Kang, J. B. et al. Nat Commun 2021) query mapping, indicative of an earlier stage of differentiation arrest ( Figure 1A bottom). Pathway analysis revealed an inflammatory signature in patients with high frequencies of stem-like cells suggestive of an inflammatory microenvironment akin to high-risk AML ( Lasry, A. et al. Nat Cancer 2023), which we refer to as inflammatory T-lineage ALL. Next, we mined our multiomic data to generate a comprehensive geneset for inflammatory T-lineage ALL scoring in single cells and bulk samples ( Figure 1A top). Interestingly, Olink cytokine array analysis uncovered upregulation of production of cytokines IL-1β, IL-18, and/or OSM in inflammatory T-lineage ALL samples. Thus, this comprehensive multiomic analysis identified a subset of T-lineage ALL patients characterized by early differentiation arrest and inflammatory signatures. To explore the relationship between inflammatory T-lineage status and clinical response, we utilized the ssGSEA algorithm to score activity of our inflammatory signature in bulk RNA-sequencing data from the TARGET T-lineage ALL dataset (n=265, 74/265 inflammatory) ( Liu, Y. et al. Nat Genet 2017) and the Tran et al. study (n=27, 17/27 inflammatory) ( Tran, T. H. et al. Blood Adv 2022). We found a significant association between high inflammatory ssGSEA score and elevated MRD. To uncover the inflammatory T-lineage ALL mutational landscape, we performed whole exome sequencing on our study cohort, and compiled the reported mutations in the TARGET dataset. Interestingly, inflammatory T-lineage ALL patients, as predicted by inflammatory signature activity, had a significantly higher prevalence of mutations affecting cytokine signaling ( NRAS, JAK3), chromatin remodeling genes ( EZH2), and the WT1 oncogene. These analyses demonstrate a significant relationship between inflammatory status, genomic lesions, and MRD. Finally, we demonstrate that inflammatory, but not non-inflammatory, T-lineage ALL cells are significantly more sensitive to BCL-2 inhibitor venetoclax ex vivo (Figure 1B).Overall, our study has identified a new subtype of T-lineage ALL defined by an early arrest in T-cell development, inflammatory signaling, a unique mutational landscape, sensitivity to venetoclax, and an association with adverse clinical parameters.

Picalm::MLLT10 can Identify a New Subgroup of Acute Leukemias of Ambiguous Lineage with Unique Extramedullary Disease and Treatment Response

Background: Mixed-phenotype acute leukemias (MPAL), which is included in acute leukemias of ambiguous lineage (ALAL), can be classified as four different subtypes based on recurring genetic alterations including BCR::ABL, KMT2A and ZNF384 gene rearrangement as well as BCL11B activation. The 2022 WHO classification has already mentioned PICALM::MLLT10 fusions are also enriched in MPAL but need more data. In 2022 ELN classification, PICALM::MLLT10 was identified as acute myeloid leukemia (AML) with other rare recurring translocations but not ALAL. In addition, there is no separate classification for PICALM::MLLT10 in the new ICC classification. Therefore, it is still confused whether PICALM::MLLT10 corresponds to AML or acute lymphoblastic leukemia (ALL) or ALAL. Methods: Fourteen PICALM::MLLT10 positive patients of 390 AL patients (14/390, 3.6%) were identified by RNA sequencing (RNA-seq) in our center from 2020-2022, including 8 female and 6 male, with a median age of 33 years (16-50 years). These patients were newly diagnosed AL according to bone marrow morphology and immunology including 5 ALAL, 5 T-ALL [ 4 early T-cell precursor ALL (ETP-ALL), 1 cortical T-ALL], 2 AML, 1 B-ALL with aberrant expression of myeloid antigen, and 1 B/T MPAL. Results: The mean white blood cell counts of these patients was 8.25×10 9/L (2.34-51×10 9/L) and platelet counts was 168.5×10 12/L (39-429×10 12/L) individually. It should be mentioned that extramedullary disease (EMD) was found in 7 cases (7/14), including mediastinum, tonsil, and skin. In terms of immunotyping, CD7 was identified in all patients (14/14) and CD33 in 71.4% (10/14) patients. The major concurrence mutations were PHF6 mutation (8/14), JAK3 mutation (5/14), and SUZ12 mutation (4/14). Characteristic cytogenetic abnormality t(10;11)(p12.3;q14.2) was only found in three cases (3/14) (Table 1). The PICALM breakpoints are mainly concentrated in exon 17 (n=6) and exon 19 (n=8). Exon 4 (n=8), exon 6 (n=2), exon 9 (n=2) and exon 10 (n=2) are the most commonbreakpointsof MLLT10. To our knowledge, this is the first report about the breakpoints and fusion gene forms of PICALM::MLLT10. For initial treatment, these patients individually received standard ALL induction chemotherapy (VDPCP, vincristine, idarubicin, pegaspargase, cyclophosphamide, prednisone) and AML induction chemotherapy (3+7 IA regimen including idarubicin, cytarabine). The initial complete remission (CR) rate was only 35.7% (5/14), and 9 patients showing no remission (NR). These NR patients subsequently received salvage chemotherapy. It is worth mentioning that 6 of them received combined chemotherapy regimen including low doses of cytarabine (LDAC), granulocyte colony-stimulating factor (G-CSF) and anthracyclines such as aclarubicin or idarubicin or homoharringtonine (CAG or IAG or HAG), and 2 of 6 patients also further received combination therapy with venetoclax (CAG or IAG +VEN). Finally, 5 of them dramatically achieved CR (5/6). In the other 3 patients, 1 patient who was failure to initial Hyper-CVAD A/B regimen also dramatically achieved CR after the more Hyper-CVAD A regimen combined with venetoclax, and 1 patient achieved CR while another still NR with both receiving CLAG (cladribine, cytarabine, G-CSF) regimen. Subsequently 11 patients (9 CR, 2 with refractory disease) received allogenic hematopoietic stem cell transplantation (allo-HSCT), and all the CR patients survived well after HSCT (follow up 3-24 months, median 15 months) without relapse, while 2 NR patients died soon after transplantation because of severe complications (Table 2). Conclusions: Our data suggested PICALM::MLLT10 positive AL should be more appropriately recognized as an independent ALAL entity and may benefit from LDAC, G-CSF and anthracyclines combination chemotherapy as well as venetoclax. Sequential HSCT after chemotherapy combined with venetoclax may further improve long-term survival in AL patients with CR even MRD positive.

Nelarabine (NEL), Pegylated Asparginase (PEG) and Venetoclax (VEN) Incorporated to HCVAD Chemotherapy in the Frontline Treatment of Adult Patients with T-Cell Acute Lymphoblastic Leukemia/Lymphoblastic Lymphoma (T-ALL/T-LBL)

Background: The incorporation of novel agents such as NEL and PEG to frontline therapy has been associated with improved outcomes in pediatric patients (pts) with T-ALL/LBL, but has not been extensively studied in the adult pts. BCL-2 upregulation has been demonstrated in T-lymphoblasts, especially of the early T-cell precursor (ETP) ALL and addition of VEN (BCL-2 inhibitor) may be beneficial. Methods: Pts with previously untreated or minimally pre-treated T-ALL/LBL were eligible if they had an ECOG PS ≤3, creatinine ≤ 2 mg/dL, total bilirubin ≤ 2 mg/dL and ALT/AST ≤ 4 x ULN. Pts received 8 cycles (C) of HCVAD (C 1,3, 5, 7) alternating with high dose Ara-C and methotrexate (MTX) (C 2, 4, 6, 8) at approximately 3-week intervals. Two cycles of NEL (650 mg/m 2 daily x 5) were initially administered after C8 (cohort 1). Later, after a protocol amendment, they were administered after C4 and C5 (cohort 2). Subsequently, the protocol was amended to add PEG (1500 IU/m 2 capped at 3750 IU; for pts age 60 or older, dose was 1000 IU/m 2, capped at 2000 IU) on day 5 of the NEL cycles (cohort 3) and more recently, VEN 400 mg daily was added on the first 7 days of each of 8 cycles of therapy (cohort 4); this was later modified to be given for 7 days during the induction cycle to all pts and reduced to 3 days per post-induction cycle only in pts with ETP-ALL or those with persistent measurable residual disease (MRD) and all other pts did not receive VEN after C1 (cohort 5). Pts with ETP-ALL were referred for allogeneic stem cell transplant (allo-SCT) in first complete remission (CR). After the completion of the intensive phase; pts in all cohorts received 30 cycles of maintenance therapy with monthly POMP (prednisone, vincristine, MTX, prednisone) and early intensification with NEL/PEG on C6 and 7 and late intensification with MTX/PEG in C18 and HCVAD on C19. All pts received 8 intrathecal chemotherapy with MTX alternating with Ara-C and mediastinal radiation was considered in pts with bulky mediastinal disease. Results: Between 7/2007 and 12/2022, 133 pts were enrolled in the 5 study cohorts sequentially (cohort 1= 30, 2= 49, 3=17, 4=16, 5=21) (Table 1). Eighty pts (60%) had T-ALL, 52 T-LBL (39%) and 1 pt had ETP/myeloid bi-phenotypic leukemia. The median age for the entire cohort was 35 years (yrs) (range 18-78), 102 pts were male (77%), 83 pts (62%) were white and 18 pts (14%) had PS ≥2. Overall, 24 pts (18%) had an ETP phenotype and another 19 pts (14%) had a near-ETP phenotype. Six pts (5%) had central nervous system disease at diagnosis, 72 pts (54%) had mediastinal disease, 49 of 80 (61%) T-ALL pts had extramedullary disease and 20 pts (15%) were in complete remission (CR) at trial enrollment after minimal pre-enrollment therapy. Overall response (CR+CRp+CRi+PR) on trial was attained in 109/113 (95%) pts [CR= 101 (89%), CRi/CRp= 3 (3%), PR=5 (4%)]. CR/CRi/CRp was attained in 36/41 (88%) pts with ETP/near-ETP ALL and 60/64 (94%) of confirmed near-ETP ALL pts ( p=0.31). At a median follow-up of 62 months (mos), the median overall survival (OS) was not reached (NR), 3-yr OS was 71% and 5-yr OS was 64% . The 3-yr OS for pts treated with HCVAD+NEL (cohort 1+2), HCVAD+NEL+PEG (Cohort 3) and HCVAD+NEL+PEG+VEN (Cohort 4+5) were 66%, 88% and 76% respectively (Figure 1). Median OS was not reached for both VEN cohorts, while 3-yr OS was 72% in cohort 4 compared to 88% in cohort 5 ( p=0.51), with significantly shorter follow-up for the latter (33 mos vs. 15 mos). Amongst the 124 pts who had CR/CRp/CRi, the median RFS was NR and 5-yr RFS was 68%. 24 (18%) pts underwent SCT in first remission, including 20 (83%) with ETP or near-ETP ALL. The median OS was shorter in the 44 ETP/near-ETP pts compared to the 77 pts with non-ETP phenotype (71 mos vs. NR, p=0.08). 30 and 60-day mortality was 0% and 1%. 10 pts (7.5%) died in remission (3, 5 and 2 pts in cohorts 1, 2 and 4 respectively) including 1 after SCT, 4 after developing therapy-related acute myeloid leukemia and 5 from infectious complications. Conclusion: The ongoing phase 2 trial of NEL, ASP, VEN added to the HCVAD regimen shows promising long-term survival in adult pts with T-ALL/LBL with 3-yr OS of 76%-88% in pts treated with HCVAD+NEL+PEG +/- VEN. Larger prospective trials and longer follow-up are needed to demonstrate further benefit.

Identification of vulnerabilities for targeting BCL-2 family members in T-Cell Acute Lymphoblastic Leukemia

T-Cell acute lymphoblastic leukemia (T-ALL) is a disease caused by the malignant transformation of T-cell lineage progenitors. With the use of intensive chemotherapies survival rates have improved, but outcomes particularly of relapsed patients remain poor. In addition, currently used intensive chemotherapies are associated with high rates of treatment-related morbidity and mortality, emphasizing the need to develop new and improved therapies. The intrinsic apoptosis pathway, one of the key pathways controlling cell death, is dysregulated in many cancers and contributes to leukemogenesis and treatment failure. The main steps of this pathway are controlled by proteins of the B-cell lymphoma (BCL-2) family at the outer mitochondrial membrane. Hence, targeting this pathway by BH3-mimetics has emerged as an effective new treatment strategy in different cancers. Venetoclax is a selective BCL-2 inhibitor and is successfully used in CLL and AML, but heterogeneous sensitivity to venetoclax has been described in ALL and inhibitors of other BCL-2 family members including BCL-XL-selective A-1331852 and MCL-1 selective AZD5991 have been developed. Moreover, a dual inhibitor of the anti-apoptotic BCL-2 family members BCL-2 and BCL-XL (AZD4320) with its dendrimer conjugate (AZD0466) has recently shown anti-tumor activity in hematologic cancer models with manageable toxicity. The aims of this study are to evaluate susceptibilities of T-ALL to inhibitors of BCL-2 family proteins, to identify markers of response, to elucidate mechanisms of action and to assess combination effects. First, we analyzed the anti-leukemia activities of inhibitors targeting BCL-2 (venetoclax), BCL-XL (A-1331852) and MCL-1 (AZD5991) and of the dual BCL-2/BCL-XL inhibitor AZD4320 in T-ALL cell lines (N=6) and a series of patient-derived xenograft (PDX) samples (N=8). Inhibition of MCL-1 alone was not effective in the cell lines tested (EC50 >1 µM), but heterogeneous sensitivity was found in PDX samples (EC50 <1µM in 3/8 samples). In cell lines, sensitivity for BCL-2 inhibition was exclusively found in a cell line with an early T-cell precursor ALL phenotype (Loucy). Similar to cell lines, we found insensitivity for BCL-2 inhibition in all PDX samples tested (N=8). For inhibition of BCL-XL, we found heterogeneous sensitivities with 4/6 cell lines and 5/8 PDX samples showing an EC 50 <1 µM. Interestingly, BH3-profiling confirmed BCL-2 dependence in Loucy and BCL-XL dependence in the other cell lines. Importantly, dual inhibition of BCL-2 and BCL-XL showed clear responses in cell lines and PDX samples (median EC 50s 796.1 nM and 721.6 nM). Notably, sensitivity was associated with priming of the BH3-peptide BAD (r s=0.89, p=0.03), providing a marker of response. We next sought to elucidate the molecular mechanisms by which different BH3-mimetics induce cell death in T-ALL. Analyzing protein interactions of BCL-2 family members by co-immunoprecipitation studies, we found that AZD4230 acts on-target by releasing BIM from BCL-2 and BCL-XL, but with compensatory increased protein complexes of BIM and MCL-1. Accordingly, combined inhibition of BCL-2 and BCL-XL with inhibition of MCL-1 leads to release of BIM from all three anti-apoptotic proteins enabling downstream apoptosis signaling. These findings suggest that combined inhibition of BCL-2 and BCL-XL together with MCL-1 may provide synergistic benefits. To further validate these data, we applied dynamic BH3-profiling and determined dependencies of T-ALL samples on BCL-2 family members in response to BCL-2/BCL-XL inhibition. Here, we found that the MCL-1 dependence strongly increases upon BCL-2/BCL-XL inhibition, suggesting MCL-1 as a target for combined inhibition. Based on these results, we performed dose-response matrix analyses to test the effects of combined BCL-2/BCL-XL and MCL-1 inhibition. Most importantly, combined inhibition showed synergism in all cell lines and PDX samples tested (Bliss scores >10), including leukemias being insensitive to single compounds. Taken together, we found vulnerabilities of T-ALL to BCL-2 family inhibition, particularly to dual BCL-2/BCL-XL inhibition by AZD4320, and we demonstrated on-target activities. Using BH3-profiling, we identified BAD-priming as a marker of response for AZD4320 and MCL-1 dependence as a resistance mechanism that can be targeted by combination treatment, suggesting further clinical evaluation.

Prognostic Value of MRD By Flow Cytometry in Adolescent and Adult Patients with Philadelphia Chromosome-Negative Acute Lymphoblastic Leukemia Treated with Pediatric-Inspired Regimen: A Post-Hoc Analysis from a Prospective Study of China

Background Minimal residual disease (MRD) is currently considered as one of the most powerful predictors in patients with Philadelphia chromosome-negative acute lymphoblastic leukemia (Ph - ALL).The time points and thresholds for MRD monitoring in pediatric Ph - ALL have been well established. However, surveillance strategy in adult ALL has not yet been standardized. In this study, we explored the prognostic value of MRD levels and monitoring time points by flow cytometry (FCM) in adolescent and adult patients with Ph - ALL treated with pediatric-inspired regimen. Methods This analysis includes adolescent and adult patients who were treated according to a pediatric-inspired regimen revised based on Children's Oncology Group protocol (CCG-1961). This prospective study was registered at Chinese Clinical Trial Registry Website with the registration number of ChiCTR-OOC-15006328. MRD was measured using bone marrow aspirates with eight-color multiparametric FCM on day 14 of induction, at end of induction (EOI, days 29-42) , post the first consolidation (post-C1), and throughout the treatment period. MRD was defined as the percentage of lymphoblasts present in the total nucleated cells counted. MRD levels of < 0.01% and ≥ 0.01% was considered as negative and positive, respectively.Patients who were MRD-positive were further stratified into three groups according to MRD level: MRD low positive (MRD-lp), ≥ 0.01% to < 0.1%; MRD high positive (MRD-hp), ≥ 0.1% to < 1%; and MRD very high positive (MRD-vhp), ≥ 1%. Results From 2014 to 2021, a total of 415 patients with a median age of 27 (range: 14-69) years were treated. Three hundred and twelve patients presented with B-precursor ALL and 103 with T-cell ALL. With a median follow-up time of 40.8 months, the median overall survival (OS) of the cohort has not yet been reached. There were 386, 389, and 346 BM samples available for MRD evaluation on day 14 of induction, at EOI, and post-C1, respectively. Figure 1A presents the distribution of MRD status over time. Figure 1B depicts the compositions of MRD levels at EOI stratified according to disease types. Notably, Ph-like ALL and early T-cell precursor (ETP) ALL were associated with extremely lower achievements of MRD negativity at EOI compared with other types. The 5-year OS rates of patients with MRD negativity, MRD-lp, MRD-hp, and MRD-vhp at EOI were 68.0% (95% CI: 60.2%-75.8%), 44.8% (95% CI: 28.9%-60.7%), 52.4% (95% CI: 34.2%-70.6%), and 36.7% (95% CI: 24.2%-49.2%), respectively ( P < 0.001) (Figure 2A). If patients were censored at the time of hematopoietic stem cell transplantation, the 5-year OS rates of these patients were 74.9% (95% CI: 65.1%-84.7%), 39.7% (95% CI: 18.7%-60.7%), 42.3% (95% CI: 11.7%-72.9%) and 0%, respectively ( P < 0.001) (Figure 2B). Patients with a negative MRD at EOI had a more favorable OS than those with a positive MRD at EOI. There was no statistically significant difference in terms of survival time between patients with MRD-lp and MRD-hp. Further, patients with MRD-vhp had the worst survival. The relapse probabilities had a similar pattern. Patients with MRD negativity at EOI had a lower 5-year CIR than those with MRD positivity at any levels. Compared to MRD-lp and MRD-hp patients, patients with MRD-vhp experienced a significantly higher rate of early relapse. However, with a longer follow-up, the 5-year cumulative incidence of relapse (CIR) did not differ significantly among patients with a positive MRD in any of the three groups, with a rate of 47.4%, 43.8%, and 49.1% in patients with MRD-lp, MRD-hp, and MRD-vhp, respectively (Figure 2C). Multivariate analysis showed that positive post-induction MRD was one of independent predictors of both OS and Disease-free survival. Conclusion Our study confirmed that MRD negativity at EOI is an independent prognostic factor of OS in adolescent and adult patients with Ph - ALL treated with pediatric-inspired regimen. Patients with MRD-lp and MRD-hp at EOI had a lower OS and a higher relapse incidence than those with MRD negativity. Therefore, an early and deep MRD clearance is important, and even extremely low MRD levels can predict poor prognosis.

Integrated Genomic Analysis Identifies a Downstream Regulatory Element Oncogenically Activating Hematopoietically Expressed Homeobox ( HHEX) in ETP-ALL

Hematopoietically-expressed Homeobox ( HHEX) is required for the maintenance of hematopoietic stem cells (HSPCs) and common lymphoid progenitor cells. HHEX is also the second most frequent integration site in retroviral insertional mutagenesis screens of leukemias and lymphomas arising in AKXD recombinant inbred mouse strains, implying that it is an important oncogene. Enforced expression of HHEX induces T-cell acute lymphoblastic leukemias (T-ALL) in murine bone marrow transplantation models. In human studies, HHEXmRNA expression is upregulated in human T-ALL studies, especially in Early T-cell Precursor (ETP-) ALL subtypes where it is concordantly expressed with LMO2. The HHEX locus is not rearranged in ETP-ALL where it is upregulated so the mechanism of HHEX activation is not clear. We analyzed T-ALL induced by retroviral mutagenesis and found an intergenic site of frequent integration, 65kb 3‘ of the Hhex coding exons. Integrations clustered within a 3 kb genomic area that is also an open chromatin region (OCR) by ATAC-seq analysis and that induced Hhex upregulation. The syntenic human region was highly enriched for H3K27 acetylation and for occupancy by multiple transcription factors, notably GATA1 and TAL1, per ENCODE. Our prior data had shown that HHEX was a downstream target of the LMO2 complex comprised of GATA1/2, TAL1 or LYL1, and nucleated by the scaffolding protein, LIM domain binding protein 1 (LDB1). We had previously shown by ChIP-PCR that the LMO2 complex was bound to the intron 1 enhancer. Thus, we performed ChIP-exonuclease analysis of LDB1 in the human ETP-ALL model cell line, LOUCY. We confirmed its occupancy at the intron 1 enhancer but we also observed LDB1 occupancy 68kb 3‘ of the HHEX coding exons, in the exact region syntenic to the murine common insertion sites. ChIP-exo analysis allowed us to pinpoint a core element with composite GATA/E box sites that was highly conserved across multiple mammalian species. LDB1 occupancy at intron 1 and at +68kb of the HHEX locus was reminiscent of LDB1's occupancy at the beta globin locus where LDB1's occupancy and homodimerization mediates chromatin looping between composite E box/GATA sites in the locus control region (LCR) and the beta globin proximal promoters. To test whether a similar interaction was occurring at the HHEX locus, we analyzed virtual 4C (chromatin conformation capture) data. We confirmed looping between the +68kb element and the proximal promoter of HHEX in human ETP-ALL primary samples and in human HSPCs. The experiments showed that looping occurs in certain developmental contexts in HSPCs and is recapitulated in ETP-ALL and mediated by the LMO2/LDB1 protein complex. HHEX is a downstream oncogene of the LMO2/LDB1 complex and is activated through this distal +68kb regulatory element. Our studies raise the possibility that dissection of this regulatory element and interference with chromatin looping are potential therapeutic mechanisms that could disrupt oncogene expression.

ALL-389 Venetoclax in the Treatment of Relapsed Refractory Early T Cell Precursor ALL (ETP-ALL). Case Report and Review of The Literature

ALL-389 Venetoclax in the Treatment of Relapsed Refractory Early T Cell Precursor ALL (ETP-ALL). Case Report and Review of The Literature

POSTER: ALL-389 Venetoclax in the Treatment of Relapsed Refractory Early T Cell Precursor ALL (ETP-ALL). Case Report and Review of The Literature

POSTER: ALL-389 Venetoclax in the Treatment of Relapsed Refractory Early T Cell Precursor ALL (ETP-ALL). Case Report and Review of The Literature

Emerging molecular subtypes and therapies in acute lymphoblastic leukemia.

Tremendous strides have been made in the molecular and cytogenetic classification of acute lymphoblastic leukemia based on gene expression profiling data, leading to an expansion of entities in the recent International Consensus Classification (ICC) of myeloid neoplasms and acute leukemias and 2022 WHO Classification of Tumours: Haematolymphoid Tumors, 5th edition. This increased diagnostic and therapeutic complexity can be overwhelming, and this review compares nomenclature differences between the ICC and WHO 5th edition publications, compiles key features of each entity, and provides a diagnostic algorithmic approach. In covering B-lymphoblastic leukemia (B-ALL), we divided the entities into established (those present in the revised 4th edition WHO) and novel (those added to either the ICC or WHO 5th edition) groups. The established B-ALL entities include B-ALL with BCR::ABL1 fusion, BCR::ABL1-like features, KMT2A rearrangement, ETV6::RUNX1 rearrangement, high hyperdiploidy, hypodiploidy (focusing on near haploid and low hypodiploid), IGH::IL3 rearrangement, TCF3::PBX1 rearrangement, and iAMP21. The novel B-ALL entities include B-ALL with MYC rearrangement; DUX4 rearrangement; MEF2D rearrangement; ZNF384 or ZNF362 rearrangement, NUTM1 rearrangement; HLF rearrangement; UBTF::ATXN7L3/PAN3,CDX2; mutated IKZF1 N159Y; mutated PAX5 P80R; ETV6::RUNX1-like features; PAX5 alteration; mutated ZEB2 (p.H1038R)/IGH::CEBPE; ZNF384 rearranged-like; KMT2A-rearranged-like; and CRLF2 rearrangement (non-Ph-like). Classification of T-ALL is complex with some variability in how the subtypes are defined in recent literature. It was classified as early T-precursor lymphoblastic leukemia/lymphoma and T-ALL, NOS in the WHO revised 4th edition and WHO 5th edition. The ICC added an entity into early T-cell precursor ALL, BCL11B-activated, and also added provisional entities subclassified based on transcription factor families that are aberrantly activated.

Comparison of flowcytometry-based scoring system for the diagnosis of early T precursor-acute lymphoblastic leukemia.

Early T cell precursor-acute lymphoblastic leukemia (ETP-ALL) is a hematolymphoid malignancy where the blasts demonstrate T cell differentiation markers along with stem cell and myeloid antigen expression. The differential diagnosis of ETP-ALL from non-ETP ALL and mixed phenotype acute leukemia is often challenging due to its overlapping immunophenotypic picture with co-expression of myeloid antigens. In this study, we endeavored to describe the immune-phenotype profile of ETP-ALL in our patients and compared the utility of four different scoring systems for better discrimination of these entities. This retrospective analysis included 31 ETP-ALL out of 860 acute leukemia cases consecutively diagnosed at the two tertiary care centers. Flowcytometry-based immunophenotype was reviewed for all the cases, and the utility of four flow-based objective scorings was assessed for the diagnosis of ETP-ALL. Receiver operating curves were drawn to compare the different flow-based scoring systems. The prevalence of ETP-ALL was 40% (n=31/77 T-ALL) in our study group, comprised mainly of adults with a median age of 20 years. The five-marker scoring system had the maximum area under the curve, followed by the seven-marker scoring system. A cut-off of ≥2.5 was more specific (sensitivity: 91%; specificity: 100%), while a score of ≥1.5 was more sensitive but slightly less specific (sensitivity: 94%, specificity: 96%). The WHO criteria for the diagnosis of ETP-ALL should be followed across all laboratories to avoid confusion and for better treatment stratification. Flow-based scoring systems can be objectively employed for better detection of cases.

Copy Number Alterations in CDKN2A/2B and MTAP Genes Are Associated With Low MEF2C Expression in T-cell Acute Lymphoblastic Leukemia.

The molecular heterogeneity of T-cell acute lymphoblastic leukemia (T-ALL) makes this disease complex. Early T-cell precursor ALL (ETP-ALL) is a recognized subtype of T-ALL associated with a high probability of induction failure with conventional therapy. Higher expression of myocyte enhancer factor 2C (MEF2C) and the absence of a biallelic deletion (ABD) are the designated markers for the ETP-ALL. Co-deletion of the contiguous genes cyclin-dependent kinase inhibitor 2A/2B (CDKN2A/2B) and the methylthioadenosine phosphorylase (MTAP) cluster, located at 9p21.3, is another common alteration in T-ALL and confers poor response to treatment. We used real-time polymerase chain reaction (PCR) analysis to assess MEF2C mRNA expression and ABD status. Copy number alterations (CNAs) in key genes previously reported to be altered in T-ALL were assessed using multiple ligation probe amplification (MLPA). We observed that CNAs in this co-deletion cluster of CDKN2A/B and MTAP genes exhibited low MEF2C expression while ABD was associated with CNA in the Abelson murine leukemia 1 (ABL1) gene. Assessment of MEF2C expression based on immunophenotype revealed that its association with CDKN2A/2B alteration is present in non-immature immunophenotype. Additionally, ABD was associated with copy number alterations of T-cell acute lymphocytic leukemia protein 1 (TAL1), myeloblastosis (MYB), and LIM domain only 2 (LMO2) genes in immature immunophenotypes. Further, STIL::TAL1fusion was associated with low expression ofMEF2C. These associations may help explain the difficulties in assessing disease heterogeneity and the prognostic importance of 9p21.3 alterations in T-ALL.

Homoharringtonine Synergizes Strongly with Venetoclax Against the Early T-Cell Progenitor Acute Lymphoblastic Leukemia：from Bench to Bed

Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) is a distinct subtype of T-ALL with poor outcome and novel therapies are urgently needed. Venetoclax (VEN), a BCL2 inhibitor, has been approved by FDA for AML. Combination of VEN and demethylating drugs or targeted therapies such as MCL1 inhibitor showed promising effect in AML. Loucy, an ETP-ALL cell line, was also reported high expression of BCL2. But little is known about VEN combination treatment in ETP-ALL. Our data showed that homoharringtonine (HHT), a plant alkaloid, downregulated MCL1 in T-ALL. Herein, we examined the treatment effect of VEN combined with HHT on ETP-ALL using murine model and patient samples. First, we compared BCL2 levels and treatment effect of VEN alone on ETP-ALL (i.e., Loucy) and T-ALL (i.e., Jurkat, Molt4, and CCRF-CEM) cell lines. We observed higher levels of BCL2 and lower IC50 values (IC50 at 24h: 0.093 uM vs.>1uM) in Loucy vs T-ALL cells. Similar results were found in human samples, with IC50 of 0.539uM in ETP-ALL sample vs 3.165-23.25uM in T-ALL samples treated with VEN for 24h. Next, to determine whether the combination of VEN and HHT shows a synergistic effect on ETP-ALL, Loucy cells were treated with a serial concentration of VEN (range: 0.025-0.1uM) and HHT (range:10-40ng/mL) alone and in combination for 24h. VEN and HHT combination showed significant synergism in reducing cell viability, with a mean combination index (CI) of < 0.1. We observed similar results in patient samples, with mean CI lower than 0.01 indicating highly synergistic effect. To assess this in vivo, a cohort of ETP-ALL mice generated by transplanting Loucy cells into NSG mice were randomly divided into 4 groups and treated with vehicle, HHT (1 mg/kg, ip), VEN (25 mg/kg, oral gavage) or HHT+VEN combination. HHT and VEN combination significantly prolonged survival of the ETP-ALL mice compared to either drug alone or vehicle (median survival of combination vs VEN vs HHT vs control: 113.5 vs 95.5 vs 85.5 vs 69.0 days, p<0.01), suggesting a synergistic effect of HHT and VEN in vivo. To identify the mechanical basis for the synergy of VEN and HHT in ETP-ALL, we performed single-cell RNA-seq using primary patient ETP-ALL cells treated with vehicle, HHT, VEN or their combination for 24h. Based on the gene expression profile, we identified a cluster of ETP-ALL cells sensitive to HHT , and a cluster only sensitive to combination of VEN and HHT which showed higher levels of Bcl2 and Mcl1 and upregulated anti-apoptosis signal by gene set enrichment analysis (GSEA), suggesting intra-tumoral heterogeneity of ETP-ALL blasts. These results suggest that HHT or VEN alone is not sufficient to induce apoptosis in the resistant cells and combination of HHT and VEN could induce apoptosis by reducing MCL1 expression in these cells which was confirmed by western blot. Further, with the promising results from bench work and given that both HHT and VEN are approved anti-leukemia drugs, we translated these findings into clinic. The HAG (HHT, low dose Cytarabine, G-CSF priming) regimen has been widely used in China for the treatment of AML for 15 years and proved to be effective and safer than intensive chemotherapy. Based on above findings, with informed consent from patients, we applied venetoclax plus HAG(V-HAG) regimen to 3 relapsed/refractory(R/R) ETP-ALL patients and 4 newly diagnosed (ND) patients, including an 82-year-old refractory patient. CR/CRi rates after the first cycle treatment were 100%, which was significantly higher than the current induction therapy (CR rate ranging from 33.3-55.9% after the first induction cycle). Except for the 82-year-old patient who refused post-remission treatment, all the remaining 6 patients remained in remission until today. Of note, no clinical manifestations of tumor lysis syndrome were observed. The median time to absolute neutrophil count recovery of >500/μL and >1000/μL was 13 (range: 0-22) and 16 (range: 0-24) days, and to platelet count recovery of > 20,000/ μL was 16 (range: 0-21) days, indicating this regimen was relatively safe and well tolerated. In conclusion, our findings demonstrate that HHT and VEN showed synergistic effect on ETP-ALL and HHT/VEN based therapy is a promising and safe regimen in newly diagnosed and R/R ETP-ALL patients. With these promising results from bench to bedside, 2 multi-center phase Ib/II clinical trials using V-HAG in patients with R/R ETP-ALL and ND-ETP have been initiated at our institution.

Pediatric Preclinical Testing Consortium Evaluation of the MCL1 Inhibitor, AMG 176, Against Xenograft Models of Acute Lymphoblastic Leukemia

Introduction: Children diagnosed with acute lymphoblastic leukemia (ALL) experience close to a 90% likelihood of cure, however the outcome for high-risk subtypes remains poor. Evasion of apoptosis is a hallmark of cancer, with dysregulation of the BCL-2 family of proteins often reported (Delbridge et al, Nat Rev Cancer 16:99-109, 2016). The BCL-2 family consists of the anti-apoptotic MCL1, BCL-2 and BCLXL as well as pro-apoptotic proteins. AMG 176, a first-in-class MCL1 inhibitor, previously demonstrated potent apoptosis-inducing activity in the nanomolar range against cell lines and patient explants of human hematological malignancies (Caenepeel et al, Cancer Discov 8:1582-97, 2018). Furthermore, discontinuous oral administration of AMG 176 inhibited the growth of human acute myeloid leukemia and multiple myeloma tumor xenografts in vivo at well-tolerated doses. AMG 176 has entered clinical evaluation for patients with hematological malignancies. Therefore, it was of interest for the Pediatric Preclinical Testing Consortium (PPTC) to evaluate the in vivo efficacy of AMG 176 against its patient-derived xenograft (PDX) models of pediatric ALL. Methods:MCL1, BCL-2 and BCLXL mRNA expression was quantified by RNA-seq (http://pedcbioportal.kidsfirstdrc.org). For in vitro cytotoxicity assays, cells were treated with AMG 176 (10 μM - 1 pM) or DMSO control for 24 h and cell viability was determined by AlamarBlue assay and expressed as IC50values. AMG 176 was evaluated in vivo against a panel of 38 ALL PDXs in a single mouse trial format. Each PDX was inoculated via tail vein injection into one NSG mouse (1-5 x 106 cells per mouse). Engraftment and drug responses were determined by enumerating the proportion of human versus mouse CD45+ (%huCD45+) cells in the peripheral blood (PB) at weekly intervals. Treatment with AMG 176 (provided by Amgen, Inc.) began once the %huCD45+ was ≥1% in each mouse and consisted of 60 mg/kg via oral gavage 2 days on, 5 days off for 2 weeks. The experimental endpoint was pre-defined as the %huCD45+ reaching 25% in the PB. Drug efficacy was assessed by event-free survival (EFS) and stringent objective response measures (Houghton et al, Pediatr Blood Cancer 49:928-40, 2007). For the pharmacokinetic (PK) study naïve NSG mice were treated with either vehicle or AMG 176 (60 mg/kg) and groups of 3 mice harvested up to 24 h post-treatment. Plasma samples were collected for analysis of AMG 176 using acetonitrile protein precipitation and LC-MS/MS with gradient elution on a Phenomenex Kinetex XB-C18 (3.0 x 50 mm, 2.6 µm) analytical column. Results:MCL1 mRNA expression ranged from 21-282 FPKM across a panel of 90 ALL PDXs representing 6 broad ALL subtypes (B-cell precursor, BCP-ALL; Ph+-ALL; Ph-like ALL; mixed-lineage leukemia-rearranged, MLLr-ALL; T-cell ALL, T-ALL; and early T-cell precursor ALL, ETP-ALL). Thirty eight of the 90 ALL PDXs were selected for in vivo evaluation of AMG 176 activity based on their profiles of MCL1, BCL-2 and BCLXL mRNA expression. AMG 176 was well tolerated in the efficacy study, with only 3 mice experiencing >10% weight loss. Mouse EFS values ranged from 3.5 to 47.8 days from treatment initiation, however no PDX achieved an objective response (remission) with all but one PDX exhibiting progressive disease. The Ph-like ALL PDX, ALL-102 (MCL1 expression 71.8 FPKM) scored a stable disease and had the highest EFS value (47.8 days). In an effort to explain the poor in vivo activity of AMG 176 several follow-up studies were performed. First, the in vitro IC50 values elicited by the AMG-176 stock against 5 leukemia cell lines (MOLM-13, 1.6 µM; NB-4, 2.0 µM; Kasumi-1, 1.6 µM; THP-1, 1.2 µM; and U-937,1.3 µM) were comparable to published data. Second, C18 LC-MS analysis revealed major and minor peaks consistent with theoretical peaks with negligible degradation. Third, PK study results demonstrated that the AMG 176 plasma concentration peaked at 8.5 µM, one hour after administration, with an AUC of 87.7 µM*hr and t1/2 of 4.1 h. This AUC was markedly lower than that in athymic nude mice treated at the same dose (1,120 µM*hr). Conclusions: AMG 176 exhibited modest in vivo activity against a large range of pediatric ALL PDXs, with the best response defined as stable disease in a single PDX. PK data suggest that AMG 176 systemic exposures in NSG mice may not be sufficient to elicit meaningful and prolonged regressions in pediatric ALL PDXs. (Supported by NCI Grants CA199000 and CA199222)

Noncanonical β-catenin interactions promote leukemia-initiating activity in early T-cell acute lymphoblastic leukemia

AbstractT-cell acute lymphoblastic leukemia (T-ALL) is a T-cell malignancy characterized by cell subsets and enriched with leukemia-initiating cells (LICs). β-Catenin modulates LIC activity in T-ALL. However, its role in maintaining established leukemia stem cells remains largely unknown. To identify functionally relevant protein interactions of β-catenin in T-ALL, we performed coimmunoprecipitation followed by liquid chromatography–mass spectrometry. Here, we report that a noncanonical functional interaction of β-catenin with the Forkhead box O3 (FOXO3) transcription factor positively regulates LIC-related genes, including the cyclin-dependent kinase 4, which is a crucial modulator of cell cycle and tumor maintenance. We also confirm the relevance of these findings using stably integrated fluorescent reporters of β-catenin and FOXO3 activity in patient-derived xenografts, which identify minor subpopulations with enriched LIC activity. In addition, gene expression data at the single-cell level of leukemic cells of primary patients at the time of diagnosis and minimal residual disease (MRD) up to 30 days after the standard treatments reveal that the expression of β-catenin– and FOXO3-dependent genes is present in the CD82+CD117+ cell fraction, which is substantially enriched with LICs in MRD as well as in early T-cell precursor ALL. These findings highlight key functional roles for β-catenin and FOXO3 and suggest novel therapeutic strategies to eradicate aggressive cell subsets in T-ALL.

136. Rare but recurrent translocations, Clustered Genomic Aberrations in Early T-cell Precursor Acute Lymphoblastic Leukemia

Chromoanagenesis is a catastrophic process that leads to acquisition of multiple genomic aberrations in one or several regions of chromosomes. Clinically significant gene fusion transcripts have been reported due to chromoanagenesis itself. We present two cases of early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) referred November 2021 and January 2022, with immunophenotype negative for CD1a, no T-cell receptor gamma chain (TRG) rearrangement. Each had complex clustered structural and numerical aberrations, rare but recurrent chromosomal translocations reported in ETP-ALL. Bone marrow (BM) biopsy could not be performed; therefore, peripheral blood (PB) was used for analysis. Case 1: A 14-year-old male with no past medical history referred for anemia, thrombocytopenia, white blood cell (WBC) 147,000/uL, 97% blasts. Chest CT showed extensive mediastinal mass encasing the mediastinal vasculature and cerebrospinal fluid (CSF) was negative for leukemia, a t(10;11)(p12;q14.1) involving PICALM::AF10 rearrangement, complex aberrations and loss of TP53 in 70% of the cells. Clinical presentation of this translocation includes organomegaly, no central nervous system (CNS) involvement and hyperleukocytosis as seen in our case. Case 2: A previously healthy 12-year-old female was referred for hyperleukocytosis, fever and abdominal pain with WBC count 653,000/uL, a translocation of t(8;12) with ETV6::NCOA2 rearrangement, complex aberrations in 100% of cells. Case 1 has significant residual disease following induction and Case 2 is undergoing induction right now, remission status unknown. Given that clustered aberrations were detected in PB instead of BM, we propose that chromoanasynthesis is the mechanism for localized shattering and reshuffling of the chromosomal regions in these cases.

Integrated clinical genotype-phenotype characteristics of early T-cell precursor acute lymphoblastic leukemia.

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is a distinct subtype of T-ALL with a unique immunophenotype and high treatment failure rate. The molecular genetic abnormalities and their prognostic impact in ETP-ALL patients are poorly understood. The authors performed systematic analyses of the clinicopathologic features with an emphasis on molecular genetic aspects of 32 patients with ETP-ALL. The median age was 43years (range, 16-71). The blasts were positive for cytoplasmic CD3 and CD7 and negative for CD1a and CD8. Other markers expressed included CD34 (88%), CD33 (72%), CD117 (68%), CD13 (58%), CD5 (partial, 56%), CD2 (38%), CD10 (25%), CD56 (partial, 19%), and CD4 (6%). Cytogenetic analyses revealed a diploid karyotype in 10 patients, simple (1-2) abnormalities in 10 patients, and complex karyotype in 10 patients. Next-generation sequencing for 21 patients demonstrated that all had gene mutations (median, four mutations per patient). The most frequently mutated genes were WT1 (38%), NOTCH1 (29%), NRAS (29%), PHF6 (25%), TP53 (24%), ASXL1 (19%), FLT3 (19%), and IKZF1 (19%). All patients except one received multi-agent chemotherapy, and 22 patients underwent allogeneic stem cell transplantation. Thrombocytopenia, an abnormal karyotype, and TP53 mutation were associated with markedly shortened overall survival. Stem cell transplantation significantly improved overall survival. Patients with ETP-ALL often have high mutation burden with increased genomic instability. TP53 mutation was the only molecular prognostic marker and was associated with complex karyotype and greater than or equal to five mutations. These patients may benefit from stem cell transplantation, and recurrent gene mutations may be novel therapeutic markers.