Ezh2 and Runx1 Mutations Collaborate to Initiate Lympho-Myeloid Leukemia in Early Thymic Progenitors.

Lympho-myeloid restricted early thymic progenitors (ETPs) are postulated to be the cell of origin for ETP leukemias, a therapy-resistant leukemia associated with frequent co-occurrence of EZH2 and RUNX1 inactivating mutations, and constitutively activating signaling pathway mutations. In a mouse model, we demonstrate that Ezh2 and Runx1 inactivation targeted to early lymphoid progenitors causes a marked expansion of pre-leukemic ETPs, showing transcriptional signatures characteristic of ETP leukemia. Addition of a RAS-signaling pathway mutation (Flt3-ITD) results in an aggressive leukemia co-expressing myeloid and lymphoid genes, which can be established and propagated in vivo by the expanded ETPs. Both mouse and human ETP leukemias show sensitivity to BET inhibition in vitro and in vivo, which reverses aberrant gene expression induced by Ezh2 inactivation

Cancer therapy shapes the fitness landscape of clonal hematopoiesis.

Acquired mutations are pervasive across normal tissues. However, understanding of the processes that drive transformation of certain clones to cancer is limited. Here we study this phenomenon in the context of clonal hematopoiesis (CH) and the development of therapy-related myeloid neoplasms (tMNs). We find that mutations are selected differentially based on exposures. Mutations in ASXL1 are enriched in current or former smokers, whereas cancer therapy with radiation, platinum and topoisomerase II inhibitors preferentially selects for mutations in DNA damage response genes (TP53, PPM1D, CHEK2). Sequential sampling provides definitive evidence that DNA damage response clones outcompete other clones when exposed to certain therapies. Among cases in which CH was previously detected, the CH mutation was present at tMN diagnosis. We identify the molecular characteristics of CH that increase risk of tMN. The increasing implementation of clinical sequencing at diagnosis provides an opportunity to identify patients at risk of tMN for prevention strategies

Cancer therapy shapes the fitness landscape of clonal hematopoiesis.

Acquired mutations are pervasive across normal tissues. However, understanding of the processes that drive transformation of certain clones to cancer is limited. Here we study this phenomenon in the context of clonal hematopoiesis (CH) and the development of therapy-related myeloid neoplasms (tMNs). We find that mutations are selected differentially based on exposures. Mutations in ASXL1 are enriched in current or former smokers, whereas cancer therapy with radiation, platinum and topoisomerase II inhibitors preferentially selects for mutations in DNA damage response genes (TP53, PPM1D, CHEK2). Sequential sampling provides definitive evidence that DNA damage response clones outcompete other clones when exposed to certain therapies. Among cases in which CH was previously detected, the CH mutation was present at tMN diagnosis. We identify the molecular characteristics of CH that increase risk of tMN. The increasing implementation of clinical sequencing at diagnosis provides an opportunity to identify patients at risk of tMN for prevention strategies

Computational tools for CNV detection using probe-level analysis of Affymetrix SNP arrays : application to the study of CNVs in follicular lymphoma

Copy number variants (CNVs) account for both variations among normal individuals and pathogenic variations. The introduction of DNA microarrays had a significant impact on the resolution of detectable CNVs and yielded a new perspective on the submicroscopic CNVs. Oligonucleotide microarrays, such as Affymetrix SNP arrays, have been commonly used for genome-wide CNV analysis. Despite the improvements in the technology, a major concern of using microarrays is how a putative CNV is defined. A disadvantage of oligonucleotide arrays is the poor signal-to-noise ratio of the data that leads to considerable variation in reported intensity readouts. Such variation will lead to false positive and false negative results, regardless of how the data are analysed. The most common approach to circumvent this problem is looking for abrupt ratio intensity shifts in several consecutive markers (e.g., SNP probes). However this approach reduces the overall resolution and mitigates the sensitivity of detecting CNVs with fewer probes. This limitation emphasizes the importance of designing methods that can identify noisy readouts at the probe-level. The main goals of this work were to study the scale of the variability in Affymetrix SNP arrays and to develop computational tools that can improve the resolution of CNV detection. By using simulated data, it was shown that the proposed method improved the accuracy and precision of detecting CNVs with fewer probes compared to standard methods. This approach was also applied to tumor/normal pairs from 25 follicular lymphoma patients and 286 candidate CNVs were found, from which 261 (91.2%) were also seen by other array-based method(s). Importantly, from 32 novel deletions, undetected by other array-based methods, at least 15 (47%) were real based on sequence-based validation. An example of a novel discovery was a partial deletion of the extracellular domain of the KIT proto-oncogene that may lead to constitutive activation of this gene. Gain of function mutations of KIT has been previously reported in several other hematologic cancers through other mechanisms such as point mutations. In conclusion, CNV discovery contributes to our understanding of complex diseases and the methods presented here should provide means for better detection of CNVs and their interpretation.Medicine, Faculty ofMedical Genetics, Department ofGraduat

Evaluating Clonal Hematopoiesis in Tumor-Infiltrating Leukocytes in Breast Cancer and Secondary Hematologic Malignancies

Chemotherapy and radiation therapy are the foundations of adjuvant therapy for early-stage breast cancer. As a complication of cytotoxic regimens, breast cancer patients are at risk for therapy-related myeloid neoplasms (t-MNs). These t-MNs are commonly refractory to antileukemic therapies and result in poor patient outcomes. We previously demonstrated that somatic mutations in leukemia-related genes are present in the tumor-infiltrating leukocytes (TILeuks) of a subset of early breast cancers. Here, we performed genomic analysis of microdissected breast cancer tumor cells and TILeuks from seven breast cancer patients who subsequently developed leukemia. In four patients, mutations present in the leukemia were detected in breast cancer TILeuks. This finding suggests that TILeuks in the primary breast cancer may harbor the ancestor of the future leukemogenic clone. Additional research is warranted to ascertain whether infiltrating mutant TILeuks could constitute a biomarker for the development of t-MN and to determine the functional consequences of mutant TILeuks

LKB1/STK11 Is a tumor suppressor in the progression of myeloproliferative neoplasms

The myeloproliferative neoplasms (MPN) frequently progress to blast phase disease, an aggressive form of acute myeloid leukemia. To identify genes that suppress disease progression, we performed a focused CRISPR/Cas9 screen and discovered that depletion of LKB1/Stk11 led to enhanced in vitro self-renewal of murine MPN cells. Deletion of Stk11 in a mouse MPN model caused rapid lethality with enhanced fibrosis, osteosclerosis, and an accumulation of immature cells in the bone marrow, as well as enhanced engraftment of primary human MPN cells in vivo. LKB1 loss was associated with increased mitochondrial reactive oxygen species and stabilization of HIF1 alpha, and downregulation of LKB1 and increased levels of HIF1 alpha were observed in human blast phase MPN specimens. Of note, we observed strong concordance of pathways that were enriched in murine MPN cells with LKB1 loss with those enriched in blast phase MPN patient specimens, supporting the conclusion that STK11 is a tumor suppressor in the MPNs. SIGNIFICANCE: Progression of the myeloproliferative neoplasms to acute myeloid leukemia occurs in a substantial number of cases, but the genetic basis has been unclear. We discovered that loss of LKB1/STK11 leads to stabilization of HIF1 alpha and promotes disease progression. This observation provides a potential therapeutic avenue for targeting progression