Carolyn S. Grove

Targeting Chromatin Regulators Inhibits Leukemogenic Gene Expression in NPM1 Mutant Leukemia

Homeobox (HOX) proteins and the receptor tyrosine kinase FLT3 are frequently highly expressed and mutated in acute myeloid leukemia (AML). Aberrant HOX expression is found in nearly all AMLs that harbor a mutation in the Nucleophosmin (NPM1) gene, and FLT3 is concomitantly mutated in approximately 60% of these cases. Little is known about how mutant NPM1 (NPM1mut) cells maintain aberrant gene expression. Here, we demonstrate that the histone modifiers MLL1 and DOT1L control HOX and FLT3 expression and differentiation in NPM1mut AML. Using a CRISPR/Cas9 genome editing domain screen, we show NPM1mut AML to be exceptionally dependent on the menin binding site in MLL1. Pharmacologic small-molecule inhibition of the menin-MLL1 protein interaction had profound antileukemic activity in human and murine models of NPM1mut AML. Combined pharmacologic inhibition of menin-MLL1 and DOT1L resulted in dramatic suppression of HOX and FLT3 expression, induction of differentiation, and superior activity against NPM1mut leukemia. SIGNIFICANCE MLL1 and DOT1L are chromatin regulators that control HOX, MEIS1, and FLT3 expression and are therapeutic targets in NPM1mut AML. Combinatorial small-molecule inhibition has synergistic on-target activity and constitutes a novel therapeutic concept for this common AML subtype. Cancer Discov; 6(10); 1166-81. ©2016 AACR.See related commentary by Hourigan and Aplan, p. 1087This article is highlighted in the In This Issue feature, p. 1069.

Development and validation of a comprehensive genomic diagnostic tool for myeloid malignancies

The diagnosis of hematologic malignancies relies on multidisciplinary workflows involving morphology, flow cytometry, cytogenetic, and molecular genetic analyses. Advances in cancer genomics have identified numerous recurrent mutations with clear prognostic and/or therapeutic significance to different cancers. In myeloid malignancies, there is a clinical imperative to test for such mutations in mainstream diagnosis; however, progress toward this has been slow and piecemeal. Here we describe Karyogene, an integrated targeted resequencing/analytical platform that detects nucleotide substitutions, insertions/deletions, chromosomal translocations, copy number abnormalities, and zygosity changes in a single assay. We validate the approach against 62 acute myeloid leukemia, 50 myelodysplastic syndrome, and 40 blood DNA samples from individuals without evidence of clonal blood disorders. We demonstrate robust detection of sequence changes in 49 genes, including difficult-to-detect mutations such as FLT3 internal-tandem and mixed-lineage leukemia (MLL) partial-tandem duplications, and clinically significant chromosomal rearrangements including MLL translocations to known and unknown partners, identifying the novel fusion gene MLL-DIAPH2 in the process. Additionally, we identify most significant chromosomal gains and losses, and several copy neutral loss-of-heterozygosity mutations at a genome-wide level, including previously unreported changes such as homozygosity for DNMT3A R882 mutations. Karyogene represents a dependable genomic diagnosis platform for translational research and for the clinical management of myeloid malignancies, which can be readily adapted for use in other cancers.

Genome-wide transposon screening and quantitative insertion site sequencing for cancer gene discovery in mice

Transposon-mediated forward genetics screening in mice has emerged as a powerful tool for cancer gene discovery. It pinpoints cancer drivers that are difficult to find with other approaches, thus complementing the sequencing-based census of human cancer genes. We describe here a large series of mouse lines for insertional mutagenesis that are compatible with two transposon systems, PiggyBac and Sleeping Beauty, and give guidance on the use of different engineered transposon variants for constitutive or tissue-specific cancer gene discovery screening. We also describe a method for semiquantitative transposon insertion site sequencing (QiSeq). The QiSeq library preparation protocol exploits acoustic DNA fragmentation to reduce bias inherent to widely used restriction–digestion-based approaches for ligation-mediated insertion site amplification. Extensive multiplexing in combination with next-generation sequencing allows affordable ultra-deep transposon insertion site recovery in high-throughput formats within 1 week. Finally, we describe principles of data analysis and interpretation for obtaining insights into cancer gene function and genetic tumor evolution.

Loss of c-Cbl E3 ubiquitin ligase activity enhances the development of myeloid leukemia in FLT3-ITD mutant mice

Mutations in the Fms-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase (RTK) occur frequently in acute myeloid leukemia (AML), with the most common involving internal tandem duplication (ITD) within the juxtamembrane domain. Fms-like tyrosine kinase 3-ITD mutations result in a mislocalized and constitutively activated receptor, which aberrantly phosphorylates signal transducer and activator of transcription 5 (STAT5) and upregulates the expression of its target genes. c-Cbl is an E3 ubiquitin ligase that negatively regulates RTKs, including FLT3, but whether it can downregulate mislocalized FLT3-ITD remains to be resolved. To help clarify this, we combined a FLT3-ITD mutation with a loss-of-function mutation in the RING finger domain of c-Cbl that abolishes its E3 ligase activity. Mice transplanted with hematopoietic stem cells expressing both mutations rapidly develop myeloid leukemia, indicating strong cooperation between the two. Although the c-Cbl mutation was shown to cause hyperactivation of another RTK, c-Kit, it had no effect on enhancing FLT3-ITD protein levels or STAT5 activation. This indicates that c-Cbl does not downregulate FLT3-ITD and that the leukemia is driven by independent pathways involving FLT3-ITDs activation of STAT5 and mutant c-Cbls activation of other RTKs, such as c-Kit. This study highlights the importance of c-Cbls negative regulation of wild-type RTKs in suppressing FLT3-ITD-driven myeloid leukemia.

Preventing chemotherapy-induced myelosuppression by repurposing the FLT3 inhibitor quizartinib

A small-molecule tyrosine kinase inhibitor protects the bone marrow from chemotherapy. Rock-a-bye bone marrow Although chemotherapy saves the lives of many cancer patients, it is a difficult treatment that induces many major side effects, with one of the most common being myelosuppression (depletion of bone marrow cells). The consequences of myelosuppression include anemia, thrombocytopenia, and neutropenia, all of which can cause severe complications and delay subsequent courses of chemotherapy. Taylor et al. discovered that quizartinib, a tyrosine kinase inhibitor, can decrease the risk of myelosuppression during cancer treatment by transiently suppressing the proliferation of bone marrow progenitor cells. In contrast, cancer cells continue to proliferate during treatment, making them a target for chemotherapy even while the bone marrow is protected, as the authors demonstrated in mice with leukemia. We describe an approach to inhibit chemotherapy-induced myelosuppression. We found that short-term exposure of mice to the FLT3 inhibitor quizartinib induced the transient quiescence of multipotent progenitors (MPPs). This property of quizartinib conferred marked protection to MPPs in mice receiving fluorouracil or gemcitabine. The protection resulted in the rapid recovery of bone marrow and blood cellularity, thus preventing otherwise lethal myelosuppression. A treatment strategy involving quizartinib priming that protected wild-type bone marrow progenitors, but not leukemic cells, from fluorouracil provided a more effective treatment than conventional induction therapy in mouse models of acute myeloid leukemia. This strategy has the potential to be extended for use in other cancers where FLT3 inhibition does not adversely affect the effectiveness of chemotherapy. Thus, the addition of quizartinib to cancer treatment regimens could markedly improve cancer patient survival and quality of life.

Molecular synergy underlies the co-occurrence patterns and phenotype of NPM1-mutant acute myeloid leukemia.

NPM1 mutations define the commonest subgroup of acute myeloid leukemia (AML) and frequently co-occur with FLT3 internal tandem duplications (ITD) or, less commonly, NRAS or KRAS mutations. Co-occurrence of mutant NPM1 with FLT3-ITD carries a significantly worse prognosis than NPM1-RAS combinations. To understand the molecular basis of these observations, we compare the effects of the 2 combinations on hematopoiesis and leukemogenesis in knock-in mice. Early effects of these mutations on hematopoiesis show that compound Npm1cA/+;NrasG12D/+ or Npm1cA;Flt3ITD share a number of features: Hox gene overexpression, enhanced self-renewal, expansion of hematopoietic progenitors, and myeloid differentiation bias. However, Npm1cA;Flt3ITD mutants displayed significantly higher peripheral leukocyte counts, early depletion of common lymphoid progenitors, and a monocytic bias in comparison with the granulocytic bias in Npm1cA/+;NrasG12D/+ mutants. Underlying this was a striking molecular synergy manifested as a dramatically altered gene expression profile in Npm1cA;Flt3ITD , but not Npm1cA/+;NrasG12D/+ , progenitors compared with wild-type. Both double-mutant models developed high-penetrance AML, although latency was significantly longer with Npm1cA/+;NrasG12D/+ During AML evolution, both models acquired additional copies of the mutant Flt3 or Nras alleles, but only Npm1cA/+;NrasG12D/+ mice showed acquisition of other human AML mutations, including IDH1 R132Q. We also find, using primary Cas9-expressing AMLs, that Hoxa genes and selected interactors or downstream targets are required for survival of both types of double-mutant AML. Our results show that molecular complementarity underlies the higher frequency and significantly worse prognosis associated with NPM1c/FLT3-ITD vs NPM1/NRAS-G12D-mutant AML and functionally confirm the role of HOXA genes in NPM1c-driven AML.

Identification of a germline F692L drug resistance variant in cis with Flt3-internal tandem duplication in knock-in mice.

Internal tandem duplication (ITD) mutations in the juxtamembrane domain of the fms-like tyrosine kinase 3 (FLT3) gene occur in approximately one quarter of cases of acute myeloid leukemia (AML),1 are associated with constitutive activation of the kinase2 and confer a poor prognosis.3 Our understanding of the molecular consequences of these mutations has benefited from studies of bespoke mouse models.4–6 Herein we report the identification of the germline variant F692L in cis with the Flt3-ITD allele of the widely studied Flt3tm1Dgg mouse.4 As this variant is analogous to the human FLT3 F691L “gatekeeper” mutation3 we investigated this finding further. We found that primary AML cells from Npm1cA; Flt3tm1Dgg double-mutant mice are resistant to sorafenib and quizartinib (AC220), but sensitive to ponatinib. The same pattern of sensitivity was observed when we tested these tyrosine kinase inhibitors against isogenic Ba/F3 cells transfected with the murine Flt3-ITD F692Lor the reverted Flt3-ITD wild-type cDNA, confirming that Flt3-ITD F692L is responsible for recapitulating the resistance pattern of the human F691L mutation. The presence of this Flt3 tyrosine kinase inhibitor-resistant variant needs to be considered when interpreting data using this model, but also makes the model an extremely useful tool for studying tyrosine kinase inhibitor resistance.

Rapid parallel acquisition of somatic mutations after NPM1 in acute myeloid leukaemia evolution.

The steps in the progression from subclinical clonal haemopoiesis to acute myeloid leukaemia (AML) have not been defined, nor has its likelihood or latency. NPM1 mutations were not found to drive clonal haemopoiesis in haematologically normal individuals(Genovese et al, 2014; Jaiswal et al, 2014; Xie et al, 2014; McKerrell et al, 2015), highlighting their strong leukaemogenic pedigree, but making them difficult to capture at a stage prior to frank AML. As occasional cases of NPM1-mutated myelodysplastic syndrome/myeloproliferative neoplasm have been described(Caudill et al, 2006; Peng et al, 2016), we hypothesised that detailed analysis of such cases could offer insights into leukaemic evolution after the acquisition of NPM1 mutations. Here, we describe the genetic events driving rapid progression of a case of NPM1 mutant chronic myelomonocytic leukaemia (CMML) to full-blown AML. A 50-year-old woman with an eight-week history of tiredness was found to be anaemic (Hb 75 g/l) and thrombocytopenic (platelet count 116 9 10/l). Her white cell (WCC) (8 7 9 10/l) and neutrophil (5 48 9 10/l) counts were normal but she had monocytosis (1 74 9 10/l). The bone marrow aspirate was haemodilute, but trephine biopsy showed marked myeloid hyperplasia and <5% blasts (Figure S1). The karyotype was normal and a diagnosis of CMML was made. After 83 d she developed fever, abdominal discomfort and a WCC of 209 9 10/l with 82% myeloblasts. A diagnosis of AML was confirmed on bone marrow aspiration (Figure S2) and molecular testing identified NPM1 and FLT3 internal tandem duplication (ITD) mutations. Analysis of CMML DNA identified the NPM1 but not the FLT3-ITD mutation. The subsequent clinical course, summarized in Fig 1, unfortunately ended when our patient succumbed to multiplyrelapsed AML 11 months later. To understand the molecular events following acquisition of the NPM1 mutation, we performed exome sequencing [SureSelect Human Exon 50 Mb Kit baits (Agilent, Santa Cara, CA), HiSeq2000 (Illumina, San Diego, CA)] of bone marrow DNA at CMML diagnosis, AML diagnosis and first complete remission. Caveman analysis identified 43 single nucleotide variants (SNV) in AML and 62 in CMML DNA, of which 23 were shared (Tables SI and SII). Pindel analysis identified ten indels in AML and eight in CMML, of which

The Diagnostic, Prognostic, and Therapeutic Utility of Molecular Testing in a Patient with Waldenstrom’s Macroglobulinemia

The application of molecular genomics and our understanding of its clinical implications in the diagnosis, prognostication and treatment of lymphoproliferative disorders has rapidly evolved over the past few years. Of particular importance are indolent B-cell malignancies where tumour cell survival and proliferation are commonly driven by mutations involving the B-cell receptor and downstream signalling pathways. In addition, the increasing number of novel therapies and targeted agents have provided clinicians with new therapeutic options with the aim of exploiting such mutations. In this case report, we highlight one such success story involving the diagnostic impact of the MYD88L265P mutation in Waldenstrom’s macroglobulinemia (WM), its prognostic implications and effect on choice of therapy in the era of novel therapies.

MRI Changes associated with Bone Marrow Reconversion can Mimic Infiltration with Multiple Myeloma

We describe a case of a fit 40-year-old who was referred for investigations to rule out Multiple Myeloma on the basis of an abnormal bone signal on MRI scanning. Haematological investigations including a bone marrow biopsy were normal and upon extended MRI re-scanning, bone changes were identified as those of marrow reconversion and attributed to his intensive exercise regime.