Inhibition of METTL3 Results in a Cell-Intrinsic Interferon Response That Enhances Antitumor Immunity.

Therapies that enhance antitumor immunity have altered the natural history of many cancers. Consequently, leveraging nonoverlapping mechanisms to increase immunogenicity of cancer cells remains a priority. Using a novel enzymatic inhibitor of the RNA methyl-transferase METTL3, we demonstrate a global decrease in N6-methyladenosine (m6A) results in double-stranded RNA (dsRNA) formation and a profound cell-intrinsic interferon response. Through unbiased CRISPR screens, we establish dsRNA-sensing and interferon signaling are primary mediators that potentiate T-cell killing of cancer cells following METTL3 inhibition. We show in a range of immunocompetent mouse models that although METTL3 inhibition is equally efficacious to anti-PD-1 therapy, the combination has far greater preclinical activity. Using SPLINTR barcoding, we demonstrate that anti-PD-1 therapy and METTL3 inhibition target distinct malignant clones, and the combination of these therapies overcomes clones insensitive to the single agents. These data provide the mole-cular and preclinical rationale for employing METTL3 inhibitors to promote antitumor immunity in the clinic. SIGNIFICANCE: This work demonstrates that METTL3 inhibition stimulates a cell-intrinsic interferon response through dsRNA formation. This immunomodulatory mechanism is distinct from current immunotherapeutic agents and provides the molecular rationale for combination with anti-PD-1 immune-checkpoint blockade to augment antitumor immunity. This article is featured in Selected Articles from This Issue, p. 2109.

Systematic profiling of conditional degron tag technologies for target validation studies.

Conditional degron tags (CDTs) are a powerful tool for target validation that combines the kinetics and reversible action of pharmacological agents with the generalizability of genetic manipulation. However, successful design of a CDT fusion protein often requires a prolonged, ad hoc cycle of construct design, failure, and re-design. To address this limitation, we report here a system to rapidly compare the activity of five unique CDTs: AID/AID2, IKZF3d, dTAG, HaloTag, and SMASh. We demonstrate the utility of this system against 16 unique protein targets. We find that expression and degradation are highly dependent on the specific CDT, the construct design, and the target. None of the CDTs leads to efficient expression and/or degradation across all targets; however, our systematic approach enables the identification of at least one optimal CDT fusion for each target. To enable the adoption of CDT strategies more broadly, we have made these reagents, and a detailed protocol, available as a community resource.

Non-genetic determinants of malignant clonal fitness at single-cell resolution.

All cancers emerge after a period of clonal selection and subsequent clonal expansion. Although the evolutionary principles imparted by genetic intratumour heterogeneity are becoming increasingly clear1, little is known about the non-genetic mechanisms that contribute to intratumour heterogeneity and malignant clonal fitness2. Here, using single-cell profiling and lineage tracing (SPLINTR)-an expressed barcoding strategy-we trace isogenic clones in three clinically relevant mouse models of acute myeloid leukaemia. We find that malignant clonal dominance is a cell-intrinsic and heritable property that is facilitated by the repression of antigen presentation and increased expression of the secretory leukocyte peptidase inhibitor gene (Slpi), which we genetically validate as a regulator of acute myeloid leukaemia. Increased transcriptional heterogeneity is a feature that enables clonal fitness in diverse tissues and immune microenvironments and in the context of clonal competition between genetically distinct clones. Similar to haematopoietic stem cells3, leukaemia stem cells (LSCs) display heritable clone-intrinsic properties of high, and low clonal output that contribute to the overall tumour mass. We demonstrate that LSC clonal output dictates sensitivity to chemotherapy and, although high- and low-output clones adapt differently to therapeutic pressure, they coordinately emerge from minimal residual disease with increased expression of the LSC program. Together, these data provide fundamental insights into the non-genetic transcriptional processes that underpin malignant clonal fitness and may inform future therapeutic strategies.

Pharmacologic Reduction of Mitochondrial Iron Triggers a Noncanonical BAX/BAK-Dependent Cell Death.

Cancer cell metabolism is increasingly recognized as providing an exciting therapeutic opportunity. However, a drug that directly couples targeting of a metabolic dependency with the induction of cell death in cancer cells has largely remained elusive. Here we report that the drug-like small-molecule ironomycin reduces the mitochondrial iron load, resulting in the potent disruption of mitochondrial metabolism. Ironomycin promotes the recruitment and activation of BAX/BAK, but the resulting mitochondrial outer membrane permeabilization (MOMP) does not lead to potent activation of the apoptotic caspases, nor is the ensuing cell death prevented by inhibiting the previously established pathways of programmed cell death. Consistent with the fact that ironomycin and BH3 mimetics induce MOMP through independent nonredundant pathways, we find that ironomycin exhibits marked in vitro and in vivo synergy with venetoclax and overcomes venetoclax resistance in primary patient samples. SIGNIFICANCE: Ironomycin couples targeting of cellular metabolism with cell death by reducing mitochondrial iron, resulting in the alteration of mitochondrial metabolism and the activation of BAX/BAK. Ironomycin induces MOMP through a different mechanism to BH3 mimetics, and consequently combination therapy has marked synergy in cancers such as acute myeloid leukemia. This article is highlighted in the In This Issue feature, p. 587.

Factors influencing international and U.S. dentists' interest in advanced periodontal education: a pilot study.

BACKGROUND: The enrollment of international periodontal students in U.S. dental schools has been increasing in recent years. Interest in applying to a periodontics specialty program may differ between U.S and international dental school graduates. The purpose of this study is to assess, from the perspective of periodontal residents, (1) factors that interest dental students to apply to periodontics programs and (2) differences in background and interest between U.S and international graduates. METHODS: A 20-question survey was sent out electronically to periodontics residents. The survey questions were designed to obtain information on the participants' backgrounds, factors that influenced them to specialize in periodontics, and their preferred features of graduate periodontics programs. The data were analyzed using descriptive statistics for socio-demographic variables, a Wilcoxon two sample test to compare mean Likert scale scores, and Fisher's exact test for associations between comparison groups. RESULTS: Of the two hundred residents invited to participate, 28% responded. The majority of the respondents stated that interest in implantology, previous exposure to periodontal procedures, interest in improving periodontal surgery skills, a good relationship with periodontics faculty, the residency curriculum, advanced program and faculty reputation as influencing factors in selecting periodontics as specialization. The majority of international graduates have up to $50,000 dollars in student debt; by comparison, half of the domestic graduates have a debt of over $250,000 dollars (p ≤ 0.05). Working experience as a dentist was significantly greater among international residents (73%) in comparison to U.S graduates (32%). In contrast with international graduates, U.S graduates more frequent reported that good relationships with the periodontics predoctoral faculty contributed to their interest in periodontics (p ≤ 0.05). Program cost and location had a greater impact on the decision of U.S. graduates than international graduates (p ≤ 0.05). CONCLUSIONS: Overall, factors associated with personal finance and predoctoral education have a greater impact on the decision of American graduates than international graduates to pursue an advanced education in periodontics, which may influence the increased enrollment of international students.

Avadomide induces degradation of ZMYM2 fusion oncoproteins in hematologic malignancies.

Thalidomide analogs exert their therapeutic effects by binding to the CRL4CRBN E3 ubiquitin ligase, promoting ubiquitination and subsequent proteasomal degradation of specific protein substrates. Drug-induced degradation of IKZF1 and IKZF3 in B-cell malignancies demonstrates the clinical utility of targeting disease-relevant transcription factors for degradation. Here, we found that avadomide (CC-122) induces CRBN-dependent ubiquitination and proteasomal degradation of ZMYM2 (ZNF198), a transcription factor involved in balanced chromosomal rearrangements with FGFR1 and FLT3 in aggressive forms of hematologic malignancies. The minimal drug-responsive element of ZMYM2 is a zinc-chelating MYM domain and is contained in the N-terminal portion of ZMYM2 that is universally included in the derived fusion proteins. We demonstrate that avadomide has the ability to induce proteasomal degradation of ZMYM2-FGFR1 and ZMYM2-FLT3 chimeric oncoproteins, both in vitro and in vivo. Our findings suggest that patients with hematologic malignancies harboring these ZMYM2 fusion proteins may benefit from avadomide treatment.

Reversible ON- and OFF-switch chimeric antigen receptors controlled by lenalidomide.

Cell-based therapies are emerging as effective agents against cancer and other diseases. As autonomous "living drugs," these therapies lack precise control. Chimeric antigen receptor (CAR) T cells effectively target hematologic malignancies but can proliferate rapidly and cause toxicity. We developed ON and OFF switches for CAR T cells using the clinically approved drug lenalidomide, which mediates the proteasomal degradation of several target proteins by inducing interactions between the CRL4CRBN E3 ubiquitin ligase and a C2H2 zinc finger degron motif. We performed a systematic screen to identify "super-degron" tags with enhanced sensitivity to lenalidomide-induced degradation and used these degradable tags to generate OFF-switch degradable CARs. To create an ON switch, we engineered a lenalidomide-inducible dimerization system and developed split CARs that required both lenalidomide and target antigen for activation. Subtherapeutic lenalidomide concentrations controlled the effector functions of ON- and OFF-switch CAR T cells. In vivo, ON-switch split CARs demonstrated lenalidomide-dependent antitumor activity, and OFF-switch degradable CARs were depleted by drug treatment to limit inflammatory cytokine production while retaining antitumor efficacy. Together, the data showed that these lenalidomide-gated switches are rapid, reversible, and clinically suitable systems to control transgene function in diverse gene- and cell-based therapies.

Small-molecule-induced polymerization triggers degradation of BCL6.

Effective and sustained inhibition of non-enzymatic oncogenic driver proteins is a major pharmacological challenge. The clinical success of thalidomide analogues demonstrates the therapeutic efficacy of drug-induced degradation of transcription factors and other cancer targets1-3, but a substantial subset of proteins are resistant to targeted degradation using existing approaches4,5. Here we report an alternative mechanism of targeted protein degradation, in which a small molecule induces the highly specific, reversible polymerization of a target protein, followed by its sequestration into cellular foci and subsequent degradation. BI-3802 is a small molecule that binds to the Broad-complex, Tramtrack and Bric-à-brac (BTB) domain of the oncogenic transcription factor B cell lymphoma 6 (BCL6) and leads to the proteasomal degradation of BCL66. We use cryo-electron microscopy to reveal how the solvent-exposed moiety of a BCL6-binding molecule contributes to a composite ligand-protein surface that engages BCL6 homodimers to form a supramolecular structure. Drug-induced formation of BCL6 filaments facilitates ubiquitination by the SIAH1 E3 ubiquitin ligase. Our findings demonstrate that a small molecule such as BI-3802 can induce polymerization coupled to highly specific protein degradation, which in the case of BCL6 leads to increased pharmacological activity compared to the effects induced by other BCL6 inhibitors. These findings open new avenues for the development of therapeutic agents and synthetic biology.

The old and the new: DNA and RNA methylation in normal and malignant hematopoiesis.

Whilst DNA cytosine methylation is the oldest and most well-studied epigenetic modification, basking in its glory days, it may be soon overshadowed by the new kid on the block: RNA adenosine methylation. This juxtaposition is indeed superficial, and a deep exploration toward the fundamental requirements for these essential epigenetic marks provides a clear perspective on their converging and synergistic roles. The recent discovery that both of these modifications are essential for preventing inappropriate activation of the intracellular innate immune responses to endogenous transcripts has provided a lot of interest in targeting them therapeutically as a means to improve cancer immunogenicity. Here we discuss the potential physiological function for DNA and RNA methylation in normal hematopoiesis and how these pervasive epigenetic marks are exploited in cancer, and provide suggestions for future research with a focus on leveraging this knowledge to uncover novel therapeutic targets.

Restricted cell cycle is essential for clonal evolution and therapeutic resistance of pre-leukemic stem cells.

Pre-leukemic stem cells (pre-LSCs) give rise to leukemic stem cells through acquisition of additional gene mutations and are an important source of relapse following chemotherapy. We postulated that cell-cycle kinetics of pre-LSCs may be an important determinant of clonal evolution and therapeutic resistance. Using a doxycycline-inducible H2B-GFP transgene in a mouse model of T-cell acute lymphoblastic leukemia to study cell cycle in vivo, we show that self-renewal, clonal evolution and therapeutic resistance are limited to a rare population of pre-LSCs with restricted cell cycle. We show that proliferative pre-LSCs are unable to return to a cell cycle-restricted state. Cell cycle-restricted pre-LSCs have activation of p53 and its downstream cell-cycle inhibitor p21. Furthermore, absence of p21 leads to proliferation of pre-LSCs, with clonal extinction through loss of asymmetric cell division and terminal differentiation. Thus, inducing proliferation of pre-LSCs represents a promising strategy to increase cure rates for acute leukemia.

Crbn I391V is sufficient to confer in vivo sensitivity to thalidomide and its derivatives in mice.

Thalidomide and its derivatives, lenalidomide and pomalidomide, are clinically effective treatments for multiple myeloma and myelodysplastic syndrome with del(5q). These molecules lack activity in murine models, limiting investigation of their therapeutic activity or toxicity in vivo. Here, we report the development of a mouse model that is sensitive to thalidomide derivatives because of a single amino acid change in the direct target of thalidomide derivatives, cereblon (Crbn). In human cells, thalidomide and its analogs bind CRBN and recruit protein targets to the CRL4CRBN E3 ubiquitin ligase, resulting in their ubiquitination and subsequent degradation by the proteasome. We show that mice with a single I391V amino acid change in Crbn exhibit thalidomide-induced degradation of drug targets previously identified in human cells, including Ikaros (Ikzf1), Aiolos (Ikzf3), Zfp91, and casein kinase 1a1 (Ck1α), both in vitro and in vivo. We use the Crbn I391V model to demonstrate that the in vivo therapeutic activity of lenalidomide in del(5q) myelodysplastic syndrome can be explained by heterozygous expression of Ck1α in del(5q) cells. We found that lenalidomide acts on hematopoietic stem cells with heterozygous expression of Ck1α and inactivation of Trp53 causes lenalidomide resistance. We further demonstrate that Crbn I391V is sufficient to confer thalidomide-induced fetal loss in mice, capturing a major toxicity of this class of drugs. Further study of the Crbn I391V model will provide valuable insights into the in vivo efficacy and toxicity of this class of drugs.

Leptin-deficient obesity prolongs survival in a murine model of myelodysplastic syndrome.

Obesity enhances the risk of developing myelodysplastic syndromes. However, the effect of obesity on survival is unclear. Obese people present with monocytosis due to inflammatory signals emanating from obese adipose tissue. We hypothesized that obesity-induced myelopoiesis would promote the transition of myelodysplastic syndrome to acute myeloid leukemia and accelerate mortality in obesity. Obese Ob/Ob mice or their lean littermate controls received a bone marrow transplant from NUP98-HOXD13 transgenic mice, a model of myelodysplastic syndrome. The metabolic parameters of the mice were examined throughout the course of the study, as were blood leukocytes. Myeloid cells were analyzed in the bone, spleen, liver and adipose tissue by flow cytometry halfway through the disease progression and at the endpoint. Survival curves were also calculated. Contrary to our hypothesis, transplantation of NUP98-HOXD13 bone marrow into obese recipient mice significantly increased survival time compared with lean recipient controls. While monocyte skewing was exacerbated in obese mice receiving NUP98-HOXD13 bone marrow, transformation to acute myeloid leukemia was not enhanced. Increased survival of obese mice was associated with a preservation of fat mass as well as increased myeloid cell deposition within the adipose tissue, and a concomitant reduction in detrimental myeloid cell accumulation within other organs. The study herein revealed that obesity increases survival in animals with myelodysplastic syndrome. This may be due to the greater fat mass of Ob/Ob mice, which acts as a sink for myeloid cells, preventing their accumulation in other key organs, such as the liver.

Lenalidomide: deciphering mechanisms of action in myeloma, myelodysplastic syndrome and beyond.

Lenalidomide and its related 'analogues' modulate the substrate specificity of the CRL4(CRBN) E3 ubiquitin ligase complex. Polyubiquitination and subsequent proteasomal degradation of IKZF1 and IKZF3 in multiple myeloma and CK1α in del(5q) MDS has recently been linked to therapeutic efficacy of this class of compounds. Harnessing ubiquitin ligase substrate specificity, may in time facilitate the degradation of other 'undruggable' proteins and allow for separation of detrimental side effects of IMiD compounds from those associated with therapeutic efficacy.