James Downing, MD, Jinghui Zhang, PhD, Charles Mullighan, MD, St. Jude Children’s Research Hospital

Genomic Analysis of Hematological Malignancies

Acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy that occurs in children. Although more than 90% of children with ALL now survive to adulthood, those with the rarest and high-risk forms of the disease continue to have poor prognoses. Through the Pediatric Cancer Genome Project (PCGP), investigators in the Hematological Malignancies Program are identifying the genetic aberrations that cause these aggressive forms of leukemias. Here we present two studies on the genetic bases of early T-cell precursor ALL and acute megakaryoblastic leukemia.

Early T-Cell Precursor ALL Is Characterized by Activating Mutations

Early T-cell precursor ALL (ETP-ALL), which comprises 15% of all pediatric T-cell leukemias, is an aggressive disease that is typically resistant to contemporary therapies. Children with ETP-ALL have a high rate of relapse and an extremely poor prognosis (i.e., 5-year survival is approximately 20%). The genetic basis of ETP-ALL has remained elusive. Although ETP-ALL is associated with a high burden of DNA copy number aberrations, none are consistently found or suggest a unifying genetic alteration that drives this disease.

Through the efforts of the PCGP, Jinghui Zhang, PhD (Computational Biology), James R. Downing, MD (Pathology), Charles G. Mullighan, MBBS(Hons), MSc, MD (Pathology), and colleagues analyzed the whole-genome sequences of leukemic cells and matched normal DNA from 12 pediatric patients with ETP-ALL. The identified genetic mutations were confirmed in a validation cohort of 52 ETP-ALL specimens and 42 non-ETP T-lineage ALLs (T-ALL).

In the journal Nature, the investigators reported that each ETP-ALL sample carried an average of 1140 sequence mutations and 12 structural variations. Of the structural variations, 51% were breakpoints in genes with well-established roles in hematopoiesis or leukemogenesis (e.g., MLH2, SUZ12, and RUNX1). Eighty-four percent of the structural variations either caused loss of function of the gene in question or resulted in the formation of a fusion gene such as ETV6-INO80D. The ETV6 gene, which encodes a protein that is essential for hematopoiesis, is frequently mutated in leukemia. Among the DNA samples sequenced in this study, ETV6 was altered in 33% of ETP-ALL but only 10% of T-ALL cases.

Of the sequence mutations identified, approximately 41% were indel mutations (i.e., mutations that cause the insertion or deletion of nucleotides in DNA), and about 9% were nonsense mutations (i.e., point mutations that result in a shortened, nonfunctional protein product). The team identified activating mutations in the cytokine receptor and RAS-signaling pathways in about 67% of the ETP-ALL samples but only 19% of T-ALL samples. Nearly 58% of the ETP-ALL samples expressed inactivating mutations in genes that control the development of hematopoietic cells, and about 48% expressed mutations in genes that modify histones.

Whole-genome sequencing identified a diverse group of novel genetic mutations in ETP-ALL. However, the high prevalence of mutations in genes that activate cytokine receptor and RAS signaling and those that inactivate hematopoietic development and histone modification suggests a common pathogenesis for ETP-ALL. To further explore this, the team performed a detailed comparison of the gene expression profiles of ETP-ALL and normal human hematopoietic progenitors.

This analysis revealed that ETP-ALL shows little similarity to human early T-cell progenitors but instead has features related to normal hematopoietic stem cells and granulocyte macrophage progenitors. In addition, the ETP-ALL signature demonstrated enrichment for a leukemia stem cell signature associated with a poor outcome in acute myeloid leukemia (AML). Together, these data suggest that ETP-ALL is distinct from T-ALL and in fact represents a stem cell leukemia that may be better treated with therapeutic approaches that include myeloid-targeted therapies. Based on these results, new therapeutic protocols that include myeloid-directed therapies are being developed.

The CBFA2T3-GLIS2 Fusion Gene Defines an Aggressive Subtype of Acute Megakaryoblastic Leukemia in Children

Acute megakaryoblastic leukemia (AMKL) is a form of AML that accounts for 10% of pediatric AML cases and only 1% of adult AML cases. Children with AMKL can be categorized into one of two subgroups: those with Down syndrome who experience a nearly 80% survival rate, and those without Down syndrome who have a poor prognosis. The 3-year survival of children with AMKL who do not have Down syndrome is only 14% to 34% despite high-intensity chemotherapy.

Although very little known about the genetic mutations that drive this disease, a study from Dr. Downing’s group has shown that AMKL is characterized by complex chromosomal rearrangements and a high number of DNA copy number abnormalities. In a more recent study led by Dr. Downing and Tanja A. Gruber, MD, PhD (Oncology, Pathology), diagnostic leukemic blasts from 14 pediatric patients with AMKL who did not have Down syndrome were analyzed by a combination of transcriptome, exome, and whole-genome sequencing to determine the functional consequences of the chromosomal aberrations previously identified. A validation group of 34 pediatric AMKL samples and 28 adult AMKL samples was also examined.

As reported in Cancer Cell, the investigators found that 12 of 14 AMKL samples expressed chimeric transcripts encoding fusion proteins. In seven samples, an inversion in chromosome 16 resulted in the fusion of CBFA2T3, which mediates quiescence of normal hematopoietic stem cells, and GLIS2, a transcription factor that is not normally expressed in the hematopoietic system. Other chimeric transcripts were identified in five of the remaining AMKL samples that did not express the CBFA2T3-GLIS2 fusion gene. In most cases, the fusion transcripts included genes that regulate the differentiation of normal megakaryocytes and those with well-established roles in leukemogenesis. In the validation cohort, six pediatric samples expressed CBFA2T3-GLIS2, but none of the adult samples did. Of note, samples carrying CBFA2T3-GLIS2 had fewer somatic mutations than did the other AMKL samples.

Outcome data from St. Jude revealed that patients with the CBFA2T3-GLIS2 fusion had significantly worse overall survival than did those who lacked the lesion (34% vs. 89%). Also, the lack of the CBFA2T3-GLIS2 and other fusion genes in adult samples highlights the biological differences between pediatric and adult AMKL. An examination of sequencing data from cancers across various tumor types analyzed in the PCGP revealed that the CBFA2T3-GLIS2 fusion is unique to AMKL. Thus, CBFA2T3-GLIS2 may serve as a prognostic marker for exceptionally high-risk AMKL.

In vitro studies of murine hematopoietic cells and human leukemia cell lines showed that the CBFA2T3-GLIS2 fusion increased BMP signaling in hematopoietic cells, which contributes to both the enhanced self-renewal capacity of the leukemic cells and their predilection to differentiate along the megakaryocytic lineage. BMP signaling has a well-established role in the development of wing structure of Drosophila flies; thus, Stacey K. Ogden, PhD (Biochemistry), and her laboratory generated transgenic Drosophila that expressed CBFA2T3-GLIS2 to determine whether the gene fusion would alter BMP signaling in vivo. The functional consequence of the overexpression of the CBFA2T3-GLIS2 fusion and full-length GLIS2 was embryonic lethality. However, the few transgenic flies that survived to adulthood demonstrated a morphologic gain-of-function phenotype.

The team concluded that the CBFA2T3-GLIS2 fusion appears to upregulate BMP signaling in vitro and in vivo. Thus, children with AMKL may benefit from therapy that blocks the BMP-signaling pathway.