ALBERT

Description: MicroRNA (miRNA) expression is sometimes dysregulated in acute myelogenous leukemia (AML), and this dysregulation has been suggested to play a role in leukemic transformation. However, somatic mutations in miRNAs are infrequent in AML. Through whole genome or exome sequencing of 200 cases of de novo AML, The Cancer Genome Atlas (NEJM 2013) identified recurring point mutations in MIR142. Heterozygous point mutations of MIR142 were identifiedin 3 cases, and bi-allelic mutations in 1 case (total incidence of 2%). All of these mutations localized to the "seed" sequence of miR-142-3p, which is critical for determining mRNA target specificity. Accordingly, we show that mutated miR-142 is unable to suppress several well-known targets of miR-142. Surprisingly, sequencing of small non-coding RNAs in AML cases carrying MIR142 mutations showed a selective loss of miR-142-5p expression. Indeed, we provide evidence that mutant miR-142-3p is selectively loaded into the RNA-induced silencing complex (RISC), resulting in degradation of miR-142-5p. Collectively, these data show that MIR142 mutations in AML likely disrupt both miRNA-142-5p and miRNA-142-3p function. To model the effect of the loss of miR-142 on hematopoiesis, we analyzed Mir142-/- mice. Prior studies in zebrafish showed that knockdown of miR-142-3p results in reduced hematopoietic stem cells (HSCs) and impaired myelopoiesis (Fan, Blood, 2014; Lu, Cell Research, 2013). Sun et al reported impaired T-cell responses in Mir142-/- mice (Sun, JCI 2015). Here, we show that loss of miR-142 is associated with a modest increase in bone marrow and splenic neutrophils. Erythroid precursors in the bone marrow are significantly reduced with a corresponding increase in the spleen. Consistent with these data, granulocyte-macrophage progenitors (GMPs) in the bone marrow are significantly increased, while megakaryocyte-erythroid progenitors (MEP) are significantly decreased. While the total number of phenotypic HSCs (CD150+ CD48- Kit+ Sca+ lineage- cells) in the bone marrow is similar to control mice, a marked increase in the percentage of CD229- myeloid-biased HSCs was observed in Mir142-/- mice (69.4% ± 3.4) versus control mice (29.2% ± 3.3; P

Description: Mutations of MIR142 have been identified in approximately 2% of de novo AML and in 20% of diffuse large B cell lymphoma (DLBCL). In AML, all of the mutations in MIR142 localize to the seed sequence of miRNA-142-3p, which is crucial in determining its target specificity. Previously, we showed that these mutations disrupt both miRNA-142-3p and miRNA-142-5p function, suggesting that loss of MIR142 plays a role in leukemic transformation (Yao et al, ASH abstract 1254, 2015). To test this hypothesis, we first characterized hematopoiesis in Mir142-/-mice. We previously reported that loss of Mir142 results in an expansion of myeloid progenitors with impaired erythropoiesis and lymphopoiesis (Yao, ASH abstract 1254, 2015). We now extend these analyses to investigate how loss of Mir142 promotes leukemic transformation. In the TCGA AML cohort, all 4 cases with somatic mutations in MIR142 also harbored mutations in either IDH1 or IDH2. To assess the functional importance of this association, we transduced wild-type or Mir142-/-hematopoietic stem/progenitor cells (HSPCs) with a retrovirus expressing the canonical IDH2 mutation, R172K. These cells were transplanted into lethally irradiated recipients and a tumor watch established. Loss of Mir142 alone was associated with mild splenomegaly, anemia, and leukopenia, but it was not sufficient to induce AML. Consistent with a prior report (Sasaki et al, Nature 2012), expression of IDH2 R172K alone induced a myeloproliferative disorder (MPD) characterized by increased myeloid cells, anemia, and splenomegaly. Concomitant loss of Mir142 did not affect the latency or penetrance of this MPD. However, the MPD in the double mutant mice was characterized by an increased percentage of CD34+ Gr1+ myeloblasts in the bone marrow and spleen plus a more severe anemia. To assess leukemia-initiating activity, we transplanted one million splenic cells into secondary recipients. Whereas IDH2 R172K alone cells rarely engrafted, Mir142-/- x IDH2 R172K cells efficiently engrafted and produced an MPD-like phenotype. These data suggest that loss of function mutations in MIR142 cooperate with IDH1/2 mutations to induce AML, possibly by increasing leukemic cell self-renewal. We examined several putative miR-142 target genes, eventually focusing on ASH1L. ASH1L is a member of the trithorax family of histone methyltransferases that has been recently implicated in MLL-associated leukemogenesis. The 3' UTR of ASH1L contains 4 putative binding sites for miRNA-142-3p, suggesting that this miRNA is critical in its post-transcriptional regulation. Indeed, in a luciferase assay with the ASH1L 3' UTR, MIR142 overexpression decreased translation by 80 percent. Consequently, Ash1l protein levels were 3 fold higher in Mir142-/- mice bone marrow compared to control mice. Since ASH1L is a key regulator of HOX gene expression, we examined HoxA9 and HoxA10 expression in Mir142-/- hematopoietic progenitor subsets. While HoxA9 and HoxA10 expression were not different in hematopoietic stem cells, they were markedly upregulated in myeloid progenitors. For example, in granulocyte-macrophage progenitors (GMPs), HoxA9 and HoxA10 expression were increased 2.86-fold and 34.4-fold, respectively in Mir142-/- versus control cells. Likewise, in megakaryocyte-erythroid progenitors (MEPs), HoxA9 and HoxA10 expression were increased 5.3-fold and 21.4-fold. Dysregulated HoxA9 and HoxA10 expression have been implicated in enhanced self-renewal capacity, and HoxA9 overexpression has been shown to cooperate with mutant IDH1 to induce AML in mice (Chaturvedi et al, Blood 2013). Collectively, these data suggest a model in which MIR142 mutations contribute to leukemogenesis by derepressing ASH1L expression, which, in turn, increases expression of HoxA9/10 and enhances self-renewal. Inhibitors targeting ASH1L may have therapeutic benefit in AML characterized by increased HOX gene expression. Disclosures No relevant conflicts of interest to declare.

Description: Acute myeloid leukemia (AML) is an oligoclonal disease marked by specific somatic genomic alterations. While the leukemia-associated mutations and rearrangements differ between individual cases, the set of recurrently mutated genes is now largely known (Cancer Genome Atlas Research Network, NEJM 2013). Current evidence supports a model of leukemogenesis, by which leukemia-associated mutations are acquired sequentially over time in hematopoietic stem cells (HSCs). Furthermore, “pre-leukemic” HSCs, which contain only a subset of the mutations found in the dominant clone, are detectable at diagnosis (Corces-Zimmerman MR, et al., PNAS 2014; Shlush LI, et al., Nature 2014). Despite these observations, the effect of these mutations, when they first arise in healthy HSCs, is largely unknown. It is likely that these early mutations endow a selective growth advantage to the HSC resulting in detectable clonal hematopoiesis without immediately causing overt leukemia. As expected, there is evidence from studies of X-inactivation skewing that clonal hematopoiesis exists in the blood of healthy elderly individuals (Busque L, et al. Blood 2009). In a separate study, hematopoietic X-inactivation skewing in elderly individuals was associated with TET2 mutations in 10/182 cases (Busque L, et al. Nat Genet 2012). This study was only capable of detecting insertions or deletions due to the high (~1%) substitution error rate of conventional next-generation sequencing (NGS) and likely underreported the prevalence of clonal hematopoiesis harboring putative driver mutations in TET2. To further study the role of leukemia-associated single nucleotide variants in healthy hematopoiesis, we applied our validated method for targeted error-corrected sequencing (ECS). ECS uses random, single molecule indexing to overcome the inherent error rate of NGS by establishing “read families” from multiple reads generated from each unique index (Schmitt MW, et al. PNAS 2012, Kinde I, et al., PNAS 2012). A dilution series of two independent mutations with technical replicates demonstrated that ECS enables the quantitative identification of variants as rare as 1:10,000 molecules. We applied ECS to identify and quantify leukemia-associated subclones harboring mutations in TP53 exons 4-7, which is where the majority of cancer-related mutations in TP53 have been described. ECS libraries were generated from blood samples drawn from 20 healthy elderly individuals (average 75 years old). Sample multiplexing for sequencing was accomplished by tagging PCR amplicons, generated from each individual, with a different oligonucleotide barcode during library preparation. The resulting individual ECS libraries were then multiplexed and sequenced on one lane of the Illumina HiSeq 2500 platform. Sequence reads originating from the same randomly indexed molecule are aligned to each other to generate read families. First, at every position, the bases called by each sequence read are compared and a consensus base is called if there is ≥90% agreement between the reads. If there is less than 90% agreement, the consensus base is called an N. Sequencing errors are thus removed since they are not shared between different reads within a read family. Second, an error corrected consensus sequence (ECCS) is discarded if

Description: Our group (Welch, Cell 2012) previously showed that hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations with age. This produces a genetically heterogeneous HSPC population with each HSPC possessing its own unique set of mutations. Later work from our group (Xie, Nature Medicine 2014) and others (Genovese, Jaiswal, NEJM 2014) demonstrated that some these mutations may provide HSPCs with a fitness advantage, allowing them to clonally expand over time in healthy individuals. We recently published data (Wong, Nature 2015) suggesting that cytotoxic therapy can select for HSPC clones with TP53 mutations, resulting in their clonal expansion and contributing to the subsequent development of therapy-related AML/MDS. From these data, we hypothesized that the intensive cytoreductive chemotherapy used to treat AML poses a significant selection pressure on a patient's non-malignant HSPC population, favoring HSPCs with specific somatic mutations and potentially resulting in oligoclonal hematopoiesis even after elimination of the founding AML clone. To test this hypothesis, we performed enhanced exome sequencing on cryopreserved bone marrow cells from 25 adult de novo AML patients (who received a "7+3" regimen for induction of remission) at time of their initial diagnosis, at first morphologic remission (~day 30), and at long-term follow up (at first relapse or during a prolonged first remission) (Klco, JAMA, in press). In 15 patients, we observed genetic clearance of the AML founding clone at the time of first morphologic remission (defined as all AML founding clone mutations declining to a variant allele frequency (VAF) 〈 2.5%). Surprisingly, in 5 of the 15 patients exhibiting clearance of their AML founding clone, we observed a concomitant expansion of a non-malignant clonal population during cytoreductive therapy, resulting in long-lived clonal hematopoiesis. Somatic mutations harbored by these expanding hematopoietic clones were validated with a high-coverage PCR-based sequencing approach. In contrast to the studies highlighting clonal hematopoiesis in individuals unexposed to chemotherapy, patients with evidence of persistent clonal hematopoiesis after cytoreductive therapy (median age = 52.2 years) were similar in age to patients without such evidence (median age = 54.1 years). The majority of these "rising clones" harbored somatic mutations in genes frequently mutated in AML such as DNMT3A, TET2 and TP53. Using next-generation sequencing and droplet digital PCR, we determined that in all of the patients with an expanding non-malignant clone, the clone was, in fact, present in the initial AML diagnosis sample at very low VAFs (0.007-0.75%). These populations rapidly expanded with chemotherapy, comprising 13-57% of the total hematopoietic population upon its completion. In all 4 of cases with sample availability, these clones remained at an expanded level a year or more after initial chemotherapy exposure. These results suggest that certain non-malignant HSPCs, having previously acquired specific aging-related somatic mutations, may gain a competitive fitness advantage after cytoreductive therapy, expand, and persist long after the completion of chemotherapy. Two of the five patients with clonal non-leukemic hematopoiesis post-chemotherapy relapsed. In both patients, the relapsed AML clone evolved from the original AML founding clone and did not involve the non-malignant clonal population, which also persisted at relapse. Both patients re-achieved morphologic remission with salvage therapy. A post-salvage therapy bone marrow sample was available in one of the cases. Interestingly, it showed that the patient's non-malignant clonal population expanded even further with salvage therapy, eventually comprising almost 80% of the total bone marrow cells. These results show that non-malignant oligoclonal hematopoiesis is common in AML patients after cytoreductive chemotherapy, with non-malignant HSPCs carrying certain somatic mutations often gaining a fitness advantage and expanding. The long-term clinical consequences of oligoclonal hematopoiesis after cytoreductive chemotherapy are unknown but are likely to be different from oligoclonal hematopoiesis developing in healthy elderly individuals. Additional studies will be required to define the mechanisms by which certain HSPCs gain a fitness advantage after cytoreductive chemotherapy. Disclosures No relevant conflicts of interest to declare.