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Despite recent progress in identifying the genetic drivers of acute lymphoblastic leukemia (ALL), prognosis remains poor for those individuals who experience disease recurrence. Moreover, acute leukemias of ambiguous lineage lack a biologically informed framework to guide classification and therapy. These needs have driven the adoption of multiple complementary single-cell sequencing approaches to explore key issues in the biology of these leukemias, including cell of origin, developmental hierarchy and ontogeny, and the molecular heterogeneity driving pathogenesis, progression, and therapeutic responsiveness. There are multiple single-cell techniques for profiling a specific modality, including RNA, DNA, chromatin accessibility and methylation; and an expanding range of approaches for simultaneous analysis of multiple modalities. Single-cell sequencing approaches have also enabled characterization of cell-intrinsic and -extrinsic features of ALL biology. In this review we describe these approaches and highlight the extensive heterogeneity that underpins ALL gene expression, cellular differentiation, and clonal architecture throughout disease pathogenesis and treatment resistance. In addition, we discuss the importance of the dynamic interactions that occur between leukemia cells and the nonleukemia microenvironment. We discuss potential opportunities and limitations of single-cell sequencing for the study of ALL biology and treatment responsiveness.

While the PROTAC approach to targeted protein degradation greatly benefits from rational design, the discovery of molecular glue degraders currently relies on screening strategies. Here, we describe screening of a library containing 3,630 cereblon binders against a panel of 9 patient-derived cancer cell lines. This led to the discovery of SJ7590, a potent degrader of CK1α, IKZF1 and IKZF3 proteins. Through a structure-guided optimization strategy we developed SJ3149, a uniquely potent and selective CK1α degrader. The crystal structure of the CK1α+CRBN+DDB1+SJ3149 quaternary complex provided a rationale for the improved degradation properties via direct contacts between SJ3149 and CK1α. In a panel of 115 human cancer cell lines SJ3149 displayed broad antiproliferative activity. This activity profile, which showed statistically significant correlation with MDM2 inhibitor Nutlin-3a, suggests potential for the development of treatments for hematological cancers, as well as solid tumors.

PURPOSE Philadelphia-like (Ph-like) B-cell ALL is a high-risk subtype of B-cell ALL that shares a gene expression profile with Ph-positive ALL, but without a BCR::ABL1 fusion. A subgroup of these patients have fusions or rearrangements involving genes such as ABL1, ABL2, PDGFRβ, JAK2, and EPOR, some of which are potentially sensitive to tyrosine kinase inhibitors (TKIs). Prompt identification of these genetic aberrations are important for prognostication and treatment decisions. PATIENTS AND METHODS We performed a retrospective review of patients with B-cell ALL treated at MD Anderson Cancer Center to identify recurrent genetic fusions commonly seen in Ph-like ALL and focus on patients treated with TKI. RESULTS We identified 23 patients with recurrent genetic fusions commonly seen in Ph-like ALL; 14 had ABL class fusions (eight ABL1, one ABL2, and five PDGFRβ) and nine had JAK2 class fusions (five JAK2 and four EPOR). Notably, several of these fusions were cryptic by conventional cytogenetics and fluorescent in situ hybridization (FISH) assays and identified only by multiplex fusion assay. Thirteen of these 23 patients received a TKI as part of their treatment; this included ABL1 fusion (n = 8), PDGFRβ fusion (n = 4), and EPOR fusion (n = 1). All four patients with ABL1 fusions who received TKI with induction chemotherapy are alive in first remission. CONCLUSION Understanding the genomics of B-cell ALL is important for disease prognostication and for precise treatment planning. Besides conventional cytogenetics and directed FISH testing, multiplex fusion assays can help identify recurrent chromosomal translocations that are seen in patients with Ph-like ALL. Early initiation of TKI appears beneficial; larger studies are required to fully understand the benefit of TKI and to design rational combination therapies for these patients.

Childhood B-cell acute lymphoblastic leukaemia (B-ALL) is characterised by recurrent genetic abnormalities that drive risk-directed treatment strategies. Using current techniques, accurate detection of such aberrations is challenging, due to the rapidly expanding list of key genetic abnormalities. Whole genome sequencing (WGS) has the potential to revolutionise genetic testing, but requires comprehensive validation. We performed WGS on 210 childhood B-ALL samples annotated with clinical and genetic data. We devised a molecular classification system to subtype these patients based on identification of key genetic changes in tumour-normal and tumour-only analyses. This approach detected 294 subtype-defining genetic abnormalities in 96% (202/210) patients. Novel genetic variants, including fusions involving genes in the MAP kinase pathway, were identified. There was excellent concordance with standard-of-care methods and whole transcriptome sequencing (WTS). We expanded the catalogue of genetic profiles that reliably classify PAX5alt and ETV6::RUNX1-like subtypes. Our novel bioinformatic pipeline improved detection of DUX4 rearrangements (DUX4-r). We defined the excellent survival rates of DUX4-r and ETV6::RUNX1-like subtypes. Overall, we comprehensively validated that WGS provides a standalone, reliable genetic test to detect all subtype-defining genetic abnormalities in B-ALL, accurately classifying patients for risk-directed treatment stratification, while simultaneously performing as an excellent research tool to identify novel disease biomarkers.

A significant proportion of infant B-cell acute lymphoblastic leukemia (B-ALL) patients remains with a dismal prognosis due to yet undetermined mechanisms. We performed a comprehensive multicohort analysis of gene expression, gene fusions, and RNA splicing alterations to uncover molecular signatures potentially linked to the observed poor outcome. We identified 87 fusions with significant allele frequency across patients and shared functional impacts, suggesting common mechanisms across fusions. We further identified a gene expression signature that predicts high risk independently of the gene fusion background and includes the upregulation of the splicing factor SRRM1. Experiments in B-ALL cell lines provided further evidence for the role of SRRM1 on cell survival, proliferation, and invasion. Supplementary analysis revealed that SRRM1 potentially modulates splicing events associated with poor outcomes through protein-protein interactions with other splicing factors. Our findings reveal a potential convergent mechanism of aberrant RNA processing that sustains a malignant phenotype independently of the underlying gene fusion and that could potentially complement current clinical strategies in infant B-ALL. Spanish Ministerio de Ciencia, Innovación y Universidades [BIO2017-85364-R]; EMBL Australia (to A.C., M.R-S., J.L.M., E.E.); National Institutes of Health [U01 CA232563 to A.T-T.]; M.T-D. acknowledges support from the Ellen Weisberg Fund: Advancing Breakthroughs in Pediatric Cancer. This work was further supported by the Spanish Ministry of Science and Innovation [MICINN, PID2019-105201RB-I00 to J.P.C.]; Junta de Andalucía [BIO-0139]; Universidad de Córdoba-FEDER [UCO-202099901918904 to J.P.C.]; GETNE2019 Research grant (to J.P.C.); CIBERobn Fisiopatología de la Obesidad y Nutrición (CIBER is an initiative of Instituto de Salud Carlos III, co-funded by the European Union: ERDF/ESF, ‘Investing in your future’).

This document describes the method of preserving and storing solid tissue specimens for banking by the Biorepository at St. Jude Children’s Research Hospital.

Acute Lymphoblastic Leukemia (ALL), the most common childhood cancer and the second most common acute leukemia in adults, arises from clonal expansion of undifferentiated lymphoid precursor cells in bone marrow. Although the majority of childhood ALL cases harbor clonal genetic alterations of transcription factor (TF) gene mutations or rearrangements, TF alterations remain difficult therapeutic targets. Small molecule induced protein degradation is a novel strategy that can be applied to currently undruggable targets such as TF and fusion oncoproteins. In this paradigm, small molecule degraders (either PROTAC or Molecular Glue (MG)) redirect the cell’s endogenous ubiquitin proteasome system and induce ubiquitination of the target protein or non-native substrate of an E3 ligase (neosubstrate) and its subsequent proteasomal degradation. Recently, the CRBN E3 ligase modulator, CC90009 was reported to show potent antitumor activity in acute myeloid leukemia, leading to the identification of GSPT1 (G1 to S phase transition factor) as a CRBN neosubstrate. These findings suggest the potential of MGs to target unanticipated vulnerabilities in different malignancies. Here, using a structurally diverse and unique set of MGs (Molecular Glue Library (MGL)) with confirmed CRBN binding affinity, we sought to identify novel CRBN modulators through phenotypic and proteomic methods. Our screening of the MGL in a panel of representative acute leukemia cell lines, including the CRLF2-rearranged ALL cell line MHH-CALL-4 identified several active MGs with EC50 Citation Format: Fatemeh Keramatnia, Yunchao Chang, Gisele Nishiguchi, Jaeki Min, Charles Mullighan, Marcus Fischer, Zoran Rankovic, Fatemeh Keramatnia. Targeting GSPT1 by a novel cereblon E3 ligase modulator for the treatment of Acute Lymphoblastic Leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA002.

Inherited non-coding genetic variants confer significant disease susceptibility in many cancers. However, the molecular processes of by which germline variants contribute to somatic lesions are poorly understood. We performed targeted sequencing in 5,008 patients and identified a key regulatory germline variant in GATA3 strongly associated with Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL). By creating an isogenic cellular model with CRISPR-Cas9 system, we showed that this variant activated a strong enhancer that significantly upregulated GATA3 transcription, which in turn reshaped the global chromatin accessibility and 3D genome organization. Remarkably, this genotype switch induced a chromatin loop between the CRLF2 oncogene and a distal enhancer, similar to the somatically acquired super-enhancer hijacking event in patients. GATA3 genotype-related alterations in transcriptional control and 3D chromatin organization were further validated in Ph-like ALL patients. Finally, we showed that GATA3 directly regulates CRLF2 and potentiates the oncogenic effects of JAK-STAT signaling in leukemogenesis. Altogether, our results provide evidence for a novel mechanism by which a germline non-coding variant contributes to oncogene activation epigenetic regulation and 3D genome reprogramming. Citation Format: Hongbo Yang, Hui Zhang, Yu Luan, Tingting Liu, Kathryn Roberts, Mao-xiang Qian, Bo Zhang, Wenjian Yang, Virginia Perez-Andreu, Jie Xu, Sriranga lyyanki, Da Kuang, Shalini Reshmi, Julie Gastier-Foster, Colton Smith, Ching-Hon Pui, William Evans, Stephen Hunger, Stephen Hunger, Leonidas Platanias, Mary Relling, Charles Mullighan, Mignon Loh, Feng Yue, Jun Yang. Non-coding germline GATA3 variants alter chromatin topology and contribute to pathogenesis of acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2118.

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy and a leading case of childhood cancer death. The last decade has witnessed a transformation in our understanding of the genetic basis of ALL due to detailed integrative genomic profiling of large cohorts of childhood ALL. Initially using microarray based approaches, and more recently with next-generation sequencing, these studies have enabled more precise sub-classification of ALL, and have shown that each ALL entity is characterized by constellations of structural and sequence mutations that typically perturb key cellular pathways including lymphoid development, cell cycle regulation, tumor suppression, Ras- and tyrosine kinase driven signaling, and epigenetic regulation. Importantly, several of the newly identified genetic alterations have entered the clinic to improve diagnosis and risk stratification, and are being pursued as new targets for therapeutic intervention. Studies of ALL have also led the way in dissecting the subclonal heterogeneity of cancer, and have shown that individual patients commonly harbor multiple related but genetically distinct subclones, and that this genetically determined clonal heterogeneity is an important determinant of relapse. In addition, genome-wide profiling has identified inherited genetic variants that influence ALL risk. Ongoing studies are deploying detailed integrative genetic transcriptomic and epigenetic sequencing to comprehensively define the genomic landscape of ALL. This review describes the recent advances in our understanding of the genetics of ALL, with an emphasis on those alterations of key pathogenic or therapeutic importance.

Inactivation of ribosomal proteins (RP) is known to cause diseases called ribosomopathies, which are often associated with abnormal hematopoiesis and an increased risk for development of leukemia. Our laboratory has recently reported an important link between inactivation of one such ribosomal protein, Rpl22, and poor survival in T cell acute lymphoblastic leukemia (T-ALL) patients. Preliminary bioinformatic analysis shows that RPL22 is lost in ∼10% of pediatric T-ALL patients, who exhibit a more aggressive disease course. Deletion of the RPL22 locus is also enriched in the early T-cell precursor (ETP-ALL) subset of T-ALL patients, which exhibit a more aggressive disease course. Thus, RPL22 loss appears to be a marker for poor outcome in pediatric T-ALL patients. While RP inactivation has previously been linked to increased cancer risk, the mechanistic basis for this linkage remained unclear. We have recently demonstrated that inactivation of Rpl22 promotes leukemic transformation by activating NF-κB and inducing the stem cell gene, LIN28B. Nevertheless, the molecular link between Rpl22 inactivation and the induction of NF-κB activity remained unclear. We have now determined that inactivation of Rpl22 activates NF-κB signaling by exacerbating ER stress responses. Indeed, Rpl22 loss results in hyperactivation of the PERK-EIF2α-ATF4 stress pathway, which is known to activate NF-κB. We have demonstrated that knockdown of PERK using shRNA abrogates the activation of NF-κB in Rpl22 mutant cells and returns Lin28B expression levels to baseline. This reduction in Lin28B eliminates the predisposition to transformation exhibited by Rpl22 mutant cells, as evidenced by decreased formation of colonies in soft agar. Taken together, these data suggest that RPL22 inactivation may serve as a negative prognostic indicator in T-ALL and that pharmacologic targeting of ER stress pathways may represent a novel therapeutic alternative in this molecularly-defined subclass of T-ALL. Citation Format: Nehal R. Solanki-Patel, Noa Greenberg, Shu Rao, Suraj Peri, Michael Slifker, Charles Mullighan, David Wiest. Role of ribosomal protein, Rpl22 in regulation of acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2864.

Childhood acute lymphoblastic leukemia is comprised of multiple subtypes defined by recurring chromosomal alterations that are important events in leukemogenesis and are widely used in diagnosis and risk stratification, yet fail to fully explain the biology of this disease. In the last 5 years, genome-wide profiling of gene expression, structural DNA alterations and sequence variations has yielded important insights into the nature of submicroscopic genetic alterations that define novel subgroups of acute lymphoblastic leukemia (ALL) and that cooperate with known cytogenetic alterations in leukemogenesis. Importantly, several of these alterations are important determinants of risk of relapse and are potential targets for therapeutic intervention. Here, these advances and future directions in the genomic analysis of ALL are discussed.

Epigenetic dysregulation is an emerging hallmark of cancers, and the identification of recurrent somatic mutations in chromatin modifying enzymes implies a causal role for altered chromatin states in tumorigenesis. While the genomic characterization of cancers is well advanced, our current understanding of cancer epigenomes is limited. Here, we developed a high-information-content mass spectrometry approach to profile global histone modifications in human cancer cells. When applied to 115 cell lines of the Cancer Cell Line Encyclopedia, this approach identified distinct molecular chromatin signatures (MCS) that provided novel insights into their epigenetic states. One MCS cluster, characterized by high H3K27 trimethylation, strongly correlated with EZH2 gain-of-function mutations, whereas another MCS cluster provided functional epigenetic correlates of EZH2 loss-of-function mutations. A third MCS cluster was characterized by increased H3K36 dimethylation and contained several cell lines harboring NSD2 translocations. Analysis of genomic correlates of the remaining cell lines within this third cluster identified a novel NSD2 E1099K variant in acute lymphoblastic leukemia (ALL) lines. Ectopic expression of the NSD2 E1099K variant induced a MCS profile characteristic of NSD2 hyperactivation and promoted transformation. Moreover, NSD2 knockdown selectively inhibited the proliferation of cell lines harboring NSD2 mutations. Massively parallel sequencing analysis of over 1000 pediatric cancer genomes identified the same NSD2 E1099K mutation in 14% of t(12;21)[ETV6-RUNX1]-containing ALLs. Together, these findings identify NSD2 as a potential therapeutic target for pediatric ALL and provide a general framework for the functional annotation of cancer epigenomes. Citation Format: Jacob Jaffe, Yan Wang, HoMan Chan, Jinghui Zhang, Roberts Huether, Gregory Kryukov, Jordan Taylor, Min Hu, Nathan Englund, Feng Yan, Zhaofu Wang, Lei Wei, Lei Wei, Jing Ma, John Easton, William R. Sellers, Nicholas Keen, Jun Liu, Jun Liu, Charles Mullighan, Steven Carr, James Downing, Levi Garraway, Frank Stegmeier. Global chromatin profiling identifies NSD2 mutations in pediatric acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr PR01.