Mechanisms of clonal evolution in childhood acute lymphoblastic leukemia.

Childhood acute lymphoblastic leukemia (ALL) can often be traced to a pre-leukemic clone carrying a prenatal genetic lesion. Postnatally acquired mutations then drive clonal evolution toward overt leukemia. The enzymes RAG1-RAG2 and AID, which diversify immunoglobulin-encoding genes, are strictly segregated in developing cells during B lymphopoiesis and peripheral mature B cells, respectively. Here we identified small pre-BII cells as a natural subset with 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 transition from the large pre-BII cell stage to the small pre-BII cell stage was exacerbated by abnormal cytokine signaling and repetitive inflammatory stimuli. We demonstrated that AID and RAG1-RAG2 drove leukemic clonal evolution with repeated exposure to inflammatory stimuli, paralleling chronic infections in childhood.

A rapid and scalable system for studying gene function in mice using conditional RNA interference.

RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi transgenic mice remains a significant limitation. By combining optimized fluorescence-coupled miR30-based shRNAs with high efficiency ES cell targeting, we developed a fast, scalable pipeline for the production of shRNA transgenic mice. Using this system, we generated eight tet-regulated shRNA transgenic lines targeting Firefly and Renilla luciferases, Oct4 and tumor suppressors p53, p16(INK4a), p19(ARF) and APC and demonstrate potent gene silencing and GFP-tracked knockdown in a broad range of tissues in vivo. Further, using an shRNA targeting APC, we illustrate how this approach can identify predicted phenotypes and also unknown functions for a well-studied gene. In addition, through regulated gene silencing we validate APC/Wnt and p19(ARF) as potential therapeutic targets in T cell acute lymphoblastic leukemia/lymphoma and lung adenocarcinoma, respectively. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene. PAPERCLIP:

LNK/SH2B3 as a novel driver in juvenile myelomonocytic leukemia.

Mutations in five canonical Ras pathway genes (NF1, NRAS, KRAS, PTPN11 and CBL) are detected in nearly 90% of patients with juvenile myelomonocytic leukemia (JMML), a frequently fatal malignant neoplasm of early childhood. In this report, we describe seven patients diagnosed with SH2B3-mutated JMML, including five patients who were found to have initiating, loss of function mutations in the gene. SH2B3 encodes the adaptor protein LNK, a negative regulator of normal hematopoiesis upstream of the Ras pathway. These mutations were identified to be germline, somatic or a combination of both. Loss of function of LNK, which has been observed in other myeloid malignancies, results in abnormal proliferation of hematopoietic cells due to cytokine hypersensitivity and activation of the JAK/STAT signaling pathway. In vitro studies of induced pluripotent stem cell-derived JMML-like hematopoietic progenitor cells (HPCs) also demonstrated sensitivity of SH2B3- mutated HPCs to JAK inhibition. Lastly, we describe two patients with JMML and SH2B3 mutations who were treated with the JAK1/2 inhibitor ruxolitinib. This report expands the spectrum of initiating mutations in JMML and raises the possibility of targeting the JAK/STAT pathway in patients with SH2B3 mutations.

Genetic disruption of N-RasG12D palmitoylation perturbs hematopoiesis and prevents myeloid transformation in mice.

Oncogenic RAS mutations pose substantial challenges for rational drug discovery. Sequence variations within the hypervariable region of Ras isoforms underlie differential posttranslational modification and subcellular trafficking, potentially resulting in selective vulnerabilities. Specifically, inhibiting the palmitoylation/depalmitoylation cycle is an appealing strategy for treating NRAS mutant cancers, particularly as normal tissues would retain K-Ras4b function for physiologic signaling. The role of endogenous N-RasG12D palmitoylation in signal transduction, hematopoietic differentiation, and myeloid transformation is unknown, and addressing these key questions will inform efforts to develop mechanism-based therapies. To evaluate the palmitoylation/depalmitoylation cycle as a candidate drug target in an in vivo disease-relevant model system, we introduced a C181S mutation into a conditional NrasG12D "knock-in" allele. The C181S second-site amino acid substitution abrogated myeloid transformation by NrasG12D, which was associated with mislocalization of the nonpalmitoylated N-Ras mutant protein, reduced Raf/MEK/ERK signaling, and alterations in hematopoietic stem and progenitor populations. Furthermore, hematologic malignancies arising in NrasG12D/G12D,C181S compound heterozygous mice invariably acquired revertant mutations that restored cysteine 181. Together, these studies validate the palmitoylation cycle as a promising therapeutic target in NRAS mutant cancers.

In utero and early-life exposure to thirdhand smoke causes profound changes to the immune system.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Thirdhand smoke (THS) is the residual tobacco contamination that remains after the smoke clears. We investigated the effects of THS exposure in utero and during early life in a transgenic Cdkn2a knockout mouse model that is vulnerable to the development of leukemia/lymphoma. Female mice, and their offspring, were exposed from the first day of pregnancy to weaning. Plasma cytokines, body weight and hematologic parameters were measured in the offspring. To investigate THS exposure effects on the development of leukemia/lymphoma, bone marrow (BM) was collected from control and THS-exposed mice and transplanted into BM-ablated recipient mice, which were followed for tumor development for 1 year. We found that in utero and early-life THS exposure caused significant changes in plasma cytokine concentrations and in immune cell populations; changes appeared more pronounced in male mice. Spleen (SP) and BM B-cell populations were significantly lower in THS-exposed mice. We furthermore observed that THS exposure increased the leukemia/lymphoma-free survival in BM transplantation recipient mice, potentially caused by THS-induced B-cell toxicity. A trend towards increased solid tumors in irradiated mice reconstituted with THS-exposed BM stimulates the hypothesis that the immunosuppressive effects of in utero and early-life THS exposure might contribute to carcinogenesis by lowering the host defense to other toxic exposures. Our study adds to expanding evidence that THS exposure alters the immune system and that in utero and early-life developmental periods represent vulnerable windows of susceptibility for these effects.

Pml nuclear body disruption cooperates in APL pathogenesis and impairs DNA damage repair pathways in mice.

A hallmark of acute promyelocytic leukemia (APL) is altered nuclear architecture, with disruption of promyelocytic leukemia (PML) nuclear bodies (NBs) mediated by the PML-retinoic acid receptor α (RARα) oncoprotein. To address whether this phenomenon plays a role in disease pathogenesis, we generated a knock-in mouse model with NB disruption mediated by 2 point mutations (C62A/C65A) in the Pml RING domain. Although no leukemias developed in PmlC62A/C65A mice, these transgenic mice also expressing RARα linked to a dimerization domain (p50-RARα model) exhibited a doubling in the rate of leukemia, with a reduced latency period. Additionally, we found that response to targeted therapy with all-trans retinoic acid in vivo was dependent on NB integrity. PML-RARα is recognized to be insufficient for development of APL, requiring acquisition of cooperating mutations. We therefore investigated whether NB disruption might be mutagenic. Compared with wild-type cells, primary PmlC62A/C65A cells exhibited increased sister-chromatid exchange and chromosome abnormalities. Moreover, functional assays showed impaired homologous recombination (HR) and nonhomologous end-joining (NHEJ) repair pathways, with defective localization of Brca1 and Rad51 to sites of DNA damage. These data directly demonstrate that Pml NBs are critical for DNA damage responses, and suggest that Pml NB disruption is a central contributor to APL pathogenesis.

TCEA1 regulates the proliferative potential of mouse myeloid cells.

Leukemia is a malignance with complex pathogenesis and poor prognosis. Discovery of noval regulators amenable to leukemia could be of value to gain insight into the pathogenesis, diagnosis and prognosis of leukemia. Here, we conducted a large-scale shRNA library screening for functional regulators in the development of myeloid cells in primary cells. We identified eighteen candidate regulators in the primary screening. Those genes cover a wide range of cellular functions, including gene expression regulation, intracellular signaling transduction, nucleotide excision repair, cell cycle control and transcription regulation. In both primary screening and validation, shRNAs targeting Tcea1, encoding the transcription elongation factor A (SII) 1, exhibited the greatest influence on the proliferative potential of cells. Knocking down the expression of Tcea1 in the 32Dcl3 myeloid cell line led to enhanced proliferation of myeloid cells and blockage of myeloid differentiation induced by G-CSF. In addition, silence of Tcea1 inhibited apoptosis of myeloid cells. Thus, Tcea1 was identified as a gene which can influence the proliferative potential, survival and differentiation of myeloid cells. These findings have implications for how transcriptional elongation influences myeloid cell development and leukemic transformation.

Age-Related Impaired Efficacy of Bone Marrow Cell Therapy for Myocardial Infarction Reflects a Decrease in B Lymphocytes.

Treatment of myocardial infarction (MI) with bone marrow cells (BMCs) improves post-MI cardiac function in rodents. However, clinical trials of BMC therapy have been less effective. While most rodent experiments use young healthy donors, patients undergoing autologous cell therapy are older and post-MI. We previously demonstrated that BMCs from aged and post-MI donor mice are therapeutically impaired, and that donor MI induces inflammatory changes in BMC composition including reduced levels of B lymphocytes. Here, we hypothesized that B cell alterations in bone marrow account for the reduced therapeutic potential of post-MI and aged donor BMCs. Injection of BMCs from increasingly aged donor mice resulted in progressively poorer cardiac function and larger infarct size. Flow cytometry revealed fewer B cells in aged donor bone marrow. Therapeutic efficacy of young healthy donor BMCs was reduced by depletion of B cells. Implantation of intact or lysed B cells improved cardiac function, whereas intact or lysed T cells provided only minor benefit. We conclude that B cells play an important paracrine role in effective BMC therapy for MI. Reduction of bone marrow B cells because of age or MI may partially explain why clinical autologous cell therapy has not matched the success of rodent experiments.

Control of the senescence-associated secretory phenotype by NF-κB promotes senescence and enhances chemosensitivity.

Cellular senescence acts as a potent barrier to tumorigenesis and contributes to the anti-tumor activity of certain chemotherapeutic agents. Senescent cells undergo a stable cell cycle arrest controlled by RB and p53 and, in addition, display a senescence-associated secretory phenotype (SASP) involving the production of factors that reinforce the senescence arrest, alter the microenvironment, and trigger immune surveillance of the senescent cells. Through a proteomics analysis of senescent chromatin, we identified the nuclear factor-κB (NF-κB) subunit p65 as a major transcription factor that accumulates on chromatin of senescent cells. We found that NF-κB acts as a master regulator of the SASP, influencing the expression of more genes than RB and p53 combined. In cultured fibroblasts, NF-κB suppression causes escape from immune recognition by natural killer (NK) cells and cooperates with p53 inactivation to bypass senescence. In a mouse lymphoma model, NF-κB inhibition bypasses treatment-induced senescence, producing drug resistance, early relapse, and reduced survival. Our results demonstrate that NF-κB controls both cell-autonomous and non-cell-autonomous aspects of the senescence program and identify a tumor-suppressive function of NF-κB that contributes to the outcome of cancer therapy.

An integrated approach to dissecting oncogene addiction implicates a Myb-coordinated self-renewal program as essential for leukemia maintenance.

Although human cancers have complex genotypes and are genomically unstable, they often remain dependent on the continued presence of single-driver mutations-a phenomenon dubbed "oncogene addiction." Such dependencies have been demonstrated in mouse models, where conditional expression systems have revealed that oncogenes able to initiate cancer are often required for tumor maintenance and progression, thus validating the pathways they control as therapeutic targets. Here, we implement an integrative approach that combines genetically defined mouse models, transcriptional profiling, and a novel inducible RNAi platform to characterize cellular programs that underlie addiction to MLL-AF9-a fusion oncoprotein involved in aggressive forms of acute myeloid leukemia (AML). We show that MLL-AF9 contributes to leukemia maintenance by enforcing a Myb-coordinated program of aberrant self-renewal involving genes linked to leukemia stem cell potential and poor prognosis in human AML. Accordingly, partial and transient Myb suppression precisely phenocopies MLL-AF9 withdrawal and eradicates aggressive AML in vivo without preventing normal myelopoiesis, indicating that strategies to inhibit Myb-dependent aberrant self-renewal programs hold promise as effective and cancer-specific therapeutics. Together, our results identify Myb as a critical mediator of oncogene addiction in AML, delineate relevant Myb target genes that are amenable to pharmacologic inhibition, and establish a general approach for dissecting oncogene addiction in vivo.

p53 loss promotes acute myeloid leukemia by enabling aberrant self-renewal.

The p53 tumor suppressor limits proliferation in response to cellular stress through several mechanisms. Here, we test whether the recently described ability of p53 to limit stem cell self-renewal suppresses tumorigenesis in acute myeloid leukemia (AML), an aggressive cancer in which p53 mutations are associated with drug resistance and adverse outcome. Our approach combined mosaic mouse models, Cre-lox technology, and in vivo RNAi to disable p53 and simultaneously activate endogenous Kras(G12D)-a common AML lesion that promotes proliferation but not self-renewal. We show that p53 inactivation strongly cooperates with oncogenic Kras(G12D) to induce aggressive AML, while both lesions on their own induce T-cell malignancies with long latency. This synergy is based on a pivotal role of p53 in limiting aberrant self-renewal of myeloid progenitor cells, such that loss of p53 counters the deleterious effects of oncogenic Kras on these cells and enables them to self-renew indefinitely. Consequently, myeloid progenitor cells expressing oncogenic Kras and lacking p53 become leukemia-initiating cells, resembling cancer stem cells capable of maintaining AML in vivo. Our results establish an efficient new strategy for interrogating oncogene cooperation, and provide strong evidence that the ability of p53 to limit aberrant self-renewal contributes to its tumor suppressor activity.

RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia.

Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention.

Crenolanib is a selective type I pan-FLT3 inhibitor.

Tyrosine kinase inhibitors (TKIs) represent transformative therapies for several malignancies. Two critical features necessary for maximizing TKI tolerability and response duration are kinase selectivity and invulnerability to resistance-conferring kinase domain (KD) mutations in the intended target. No prior TKI has demonstrated both of these properties. Aiming to maximize selectivity, medicinal chemists have largely sought to create TKIs that bind to an inactive (type II) kinase conformation. Here we demonstrate that the investigational type I TKI crenolanib is a potent inhibitor of Fms tyrosine kinase-3 (FLT3) internal tandem duplication, a validated therapeutic target in human acute myeloid leukemia (AML), as well as all secondary KD mutants previously shown to confer resistance to the first highly active FLT3 TKI quizartinib. Moreover, crenolanib is highly selective for FLT3 relative to the closely related protein tyrosine kinase KIT, demonstrating that simultaneous FLT3/KIT inhibition, a prominent feature of other clinically active FLT3 TKIs, is not required for AML cell cytotoxicity in vitro and may contribute to undesirable toxicity in patients. A saturation mutagenesis screen of FLT3-internal tandem duplication failed to recover any resistant colonies in the presence of a crenolanib concentration well below what has been safely achieved in humans, suggesting that crenolanib has the potential to suppress KD mutation-mediated clinical resistance. Crenolanib represents the first TKI to exhibit both kinase selectivity and invulnerability to resistance-conferring KD mutations, which is unexpected of a type I inhibitor. Crenolanib has significant promise for achieving deep and durable responses in FLT3-mutant AML, and may have a profound impact upon future medicinal chemistry efforts in oncology.

Mouse models of human AML accurately predict chemotherapy response.

The genetic heterogeneity of cancer influences the trajectory of tumor progression and may underlie clinical variation in therapy response. To model such heterogeneity, we produced genetically and pathologically accurate mouse models of common forms of human acute myeloid leukemia (AML) and developed methods to mimic standard induction chemotherapy and efficiently monitor therapy response. We see that murine AMLs harboring two common human AML genotypes show remarkably diverse responses to conventional therapy that mirror clinical experience. Specifically, murine leukemias expressing the AML1/ETO fusion oncoprotein, associated with a favorable prognosis in patients, show a dramatic response to induction chemotherapy owing to robust activation of the p53 tumor suppressor network. Conversely, murine leukemias expressing MLL fusion proteins, associated with a dismal prognosis in patients, are drug-resistant due to an attenuated p53 response. Our studies highlight the importance of genetic information in guiding the treatment of human AML, functionally establish the p53 network as a central determinant of chemotherapy response in AML, and demonstrate that genetically engineered mouse models of human cancer can accurately predict therapy response in patients.

Preclinical efficacy of MEK inhibition in Nras-mutant AML.

Oncogenic NRAS mutations are highly prevalent in acute myeloid leukemia (AML). Genetic analysis supports the hypothesis that NRAS mutations cooperate with antecedent molecular lesions in leukemogenesis, but have limited independent prognostic significance. Using short hairpin RNA-mediated knockdown in human cell lines and primary mouse leukemias, we show that AML cells with NRAS/Nras mutations are dependent on continued oncogene expression in vitro and in vivo. Using the Mx1-Cre transgene to inactivate a conditional mutant Nras allele, we analyzed hematopoiesis and hematopoietic stem and progenitor cells (HSPCs) under normal and stressed conditions and found that HSPCs lacking Nras expression are functionally equivalent to normal HSPCs in the adult mouse. Treating recipient mice transplanted with primary Nras(G12D) AMLs with 2 potent allosteric mitogen-activated protein kinase kinase (MEK) inhibitors (PD0325901 or trametinib/GlaxoSmithKline 1120212) significantly prolonged survival and reduced proliferation but did not induce apoptosis, promote differentiation, or drive clonal evolution. The phosphatidylinositol 3-kinase inhibitor GDC-0941 was ineffective as a single agent and did not augment the activity of PD0325901. All mice ultimately succumbed to progressive leukemia. Together, these data validate oncogenic N-Ras signaling as a therapeutic target in AML and support testing combination regimens that include MEK inhibitors.

Co-operative leukemogenesis in acute myeloid leukemia and acute promyelocytic leukemia reveals C/EBPα as a common target of TRIB1 and PML/RARA.

The PML/RARA fusion protein occurs as a result of the t(15;17) translocation in the acute promyelocytic leukemia subtype of human acute myeloid leukemia. Gain of chromosome 8 is the most common chromosomal gain in human acute myeloid leukemia, including acute promyelocytic leukemia. We previously demonstrated that gain of chromosome 8-containing MYC is of central importance in trisomy 8, but the role of the nearby TRIB1 gene has not been experimentally addressed in this context. We have now tested the hypothesis that both MYC and TRIB1 have functional roles underlying leukemogenesis of trisomy 8 by using retroviral vectors to express MYC and TRIB1 in wild-type bone marrow and in marrow that expressed a PML/RARA transgene. Interestingly, although MYC and TRIB1 readily co-operated in leukemogenesis for wild-type bone marrow, TRIB1 provided no selective advantage to cells expressing PML/RARA. We hypothesized that this lack of co-operation between PML/RARA and TRIB1 reflected a common pathway for their effect: both proteins targeting the myeloid transcription factor C/EBPα. In support of this idea, TRIB1 expression abrogated the all-trans retinoic acid response of acute promyelocytic leukemia cells in vitro and in vivo Our data delineate the common and redundant inhibitory effects of TRIB1 and PML/RARA on C/EBPα providing a potential explanation for the lack of selection of TRIB1 in human acute promyelocytic leukemia, and highlighting the key role of C/EBPs in acute promyelocytic leukemia pathogenesis and therapeutic response. In addition, the co-operativity we observed between MYC and TRIB1 in the absence of PML/RARA show that, outside of acute promyelocytic leukemia, gain of both genes may drive selection for trisomy 8.