Here a new, intrinsically pluripotent, CD45-negative population from human cord blood, termed unrestricted somatic stem cells (USSCs) is described. This rare population grows adherently and can be expanded to 1015 cells without losing pluripotency. In vitro USSCs showed homogeneous differentiation into osteoblasts, chondroblasts, adipocytes, and hematopoietic and neural cells including astrocytes and neurons that express neurofilament, sodium channel protein, and various neurotransmitter phenotypes. Stereotactic implantation of USSCs into intact adult rat brain revealed that human Tau-positive cells persisted for up to 3 mo and showed migratory activity and a typical neuron-like morphology. In vivo differentiation of USSCs along mesodermal and endodermal pathways was demonstrated in animal models. Bony reconstitution was observed after transplantation of USSC-loaded calcium phosphate cylinders in nude rat femurs. Chondrogenesis occurred after transplanting cell-loaded gelfoam sponges into nude mice. Transplantation of USSCs in a noninjury model, the preimmune fetal sheep, resulted in up to 5% human hematopoietic engraftment. More than 20% albumin-producing human parenchymal hepatic cells with absence of cell fusion and substantial numbers of human cardiomyocytes in both atria and ventricles of the sheep heart were detected many months after USSC transplantation. No tumor formation was observed in any of these animals.
B-cell leukemia/lymphoma 2 (BCL-2) prevents commitment to programmed cell death at the mitochondrion. It remains a challenge to identify those tumors that are best treated by inhibition of BCL-2. Here we demonstrate that acute myeloid leukemia (AML) cell lines, primary patient samples, and murine primary xenografts are very sensitive to treatment with the selective BCL-2 antagonist ABT-199. In primary patient cells, the median IC50 was approximately 10 nM, and cell death occurred within 2 h. Our ex vivo sensitivity results compare favorably with those observed for chronic lymphocytic leukemia (CLL), a disease for which ABT-199 has demonstrated consistent activity in clinical trials. Moreover, mitochondrial studies using BH3 profiling demonstrate activity at the mitochondrion that correlates well with cytotoxicity, supporting an on target mitochondrial mechanism of action. Our protein and BH3 profiling studies provide promising tools that can be tested as predictive biomarkers in any clinical trial of ABT-199 in AML.
The ETV6-RUNX1 fusion gene, found in 25% of childhood acute lymphoblastic leukemia (ALL), is acquired in utero but requires additional somatic mutations for overt leukemia. We used exome and low-coverage whole-genome sequencing to characterize secondary events associated with leukemic transformation. RAG-mediated deletions emerge as the dominant mutational process, characterized by recombination signal sequence motifs near the breakpoints; incorporation of non-templated sequence at the junction; ~30-fold enrichment at promoters and enhancers of genes actively transcribed in B-cell development and an unexpectedly high ratio of recurrent to non-recurrent structural variants. Single cell tracking shows that this mechanism is active throughout leukemic evolution with evidence of localized clustering and re-iterated deletions. Integration of point mutation and rearrangement data identifies ATF7IP and MGA as two new tumor suppressor genes in ALL. Thus, a remarkably parsimonious mutational process transforms ETV6-RUNX1 lymphoblasts, targeting the promoters, enhancers and first exons of genes that normally regulate B-cell differentiation.
SUMMARY We have assembled, annotated, and analyzed a database of over 1700 breakpoints from the most common chromosomal rearrangements in human leukemias and lymphomas. Using this database, we show that although the CpG dinucleotide constitutes only 1% of the human genome, it accounts for 40–70% of breakpoints at proB/pre-B stage translocation regions – specifically, those near the bcl-2, bcl-1, and E2A genes. We do not observe CpG hotspots in rearrangements involving lymphoid-myeloid progenitors, mature B cells, or T cells. The stage-specificity, lineage-specificity, CpG targeting, and unique breakpoint distributions at these cluster regions may be explained by a lesion-specific double-strand breakage mechanism involving the RAG complex acting at AID-deaminated methyl-CpGs.
Tyrosine kinase inhibitors (TKI) are widely used to treat patients with leukemia driven by BCR-ABL11 and other oncogenic tyrosine kinases2,3. Recent efforts focused on the development of more potent TKI that also inhibit mutant tyrosine kinases4,5. However, even effective TKI typically fail to eradicate leukemia-initiating cells6–8, which often cause recurrence of leukemia after initially successful treatment. Here we report on the discovery of a novel mechanism of drug-resistance, which is based on protective feedback signaling of leukemia cells in response to TKI-treatment. We identified BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukemia-initiating subclones. BCL6 is a known proto-oncogene that is often translocated in diffuse large B cell lymphoma (DLBCL)9. In response to TKI-treatment, BCR-ABL1 acute lymphoblastic leukemia (ALL) cells upregulate BCL6 protein levels by ~90-fold, i.e. to similar levels as in DLBCL (Fig. 1a). Upregulation of BCL6 in response to TKI-treatment represents a novel defense mechanism, which enables leukemia cells to survive TKI-treatment: Previous work suggested that TKI-mediated cell death is largely p53-independent. Here we demonstrate that BCL6 upregulation upon TKI-treatment leads to transcriptional inactivation of the p53 pathway. BCL6-deficient leukemia cells fail to inactivate p53 and are particularly sensitive to TKI-treatment. BCL6−/− leukemia cells are poised to undergo cellular senescence and fail to initiate leukemia in serial transplant recipients. A combination of TKI-treatment and a novel BCL6 peptide inhibitor markedly increased survival of NOD/SCID mice xenografted with patient-derived BCR-ABL1 ALL cells. We propose that dual targeting of oncogenic tyrosine kinases and BCL6-dependent feedback (Supplementary Fig. 1) represents a novel strategy to eradicate drug-resistant and leukemia-initiating subclones in tyrosine kinase-driven leukemia.
SUMMARY We report that t(1;19)-ALL cells universally exhibit expression of and dependence on the cell surface receptor, ROR1. We further identify t(1;19)-ALL cell sensitivity to the kinase inhibitor dasatinib due to its inhibition of the pre-B-cell receptor (pre-BCR) signaling complex. These phenotypes are a consequence of developmental arrest at an intermediate/late stage of B-lineage maturation. Additionally, inhibition of pre-BCR signaling induces further ROR1 upregulation, and we identify distinct ROR1 and pre-BCR downstream signaling pathways that are modulated in a counter-balancing manner—both leading to AKT phosphorylation. Consistent with this, AKT phosphorylation is transiently eliminated after dasatinib treatment, but is partially restored following dasatinib potentiation of ROR1 expression. Consequently, ROR1 silencing accentuates dasatinib killing of t(1;19)-ALL cells.
SUMMARY Studying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR+ ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR+ ALL.
Childhood acute lymphoblastic leukemia can often be retraced to a pre-leukemic clone carrying a prenatal genetic lesion. Postnatally acquired mutations then drive clonal evolution towards overt leukemia. RAG1-RAG2 and AID enzymes, the diversifiers of immunoglobulin genes, are strictly segregated to early and late stages of B-lymphopoiesis, respectively. Here, we identified small pre-BII cells as a natural subset of increased genetic vulnerability owing to concurrent activation of these enzymes. Consistent with epidemiological findings on childhood ALL etiology, susceptibility to genetic lesions during B-lymphopoiesis at the large to small pre-BII transition is exacerbated by abnormal cytokine signaling and repetitive inflammatory stimuli. We demonstrate that AID and RAG1-RAG2 drive leukemic clonal evolution with repeated exposure to inflammatory stimuli, paralleling chronic infections in childhood.
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