ALBERT

Description: Targeted inhibitors to oncogenic kinases demonstrate encouraging clinical responses early in the treatment course, however, most patients will relapse due to target-dependent mechanisms that mitigate enzyme-inhibitor binding or through target-independent mechanisms, such as alternate activation of survival and proliferation pathways, known as adaptive resistance. One example involves the FMS-like receptor tyrosine kinase (FLT3). Activating mutations of FLT3 result in its autophosphorylation and initiation of intracellular signaling pathways, which induce abnormal survival and proliferation of leukemic cells.One of the most common mutations in acute myeloid leukemia (AML) involves the internal tandem duplication (ITD) of FLT3, which occurs in ~25% of all cases of newly diagnosed AML and confers a particularly poor prognosis. FLT3 inhibitors (FLT3i) evaluated in clinical studies as monotherapy and combination therapies have shown good initial response rates; however, patients eventually relapse with FLT3i-resistant disease. The absence of durable remission in patients treated with potent and selective FLT3i highlights the need to identify resistance mechanisms and develop additional treatment strategies. Several mechanisms contribute to resistance to selective FLT3i, including mutations in the tyrosine kinase domain of FLT3 (20-50%) or activation of parallel signaling mechanisms that bypass FLT3 signaling, referred to as adaptive resistance (30-50%). Here we describe mechanisms of adaptive resistance in FLT3-mutant AML by examining in-cell kinase and gene regulatory network responses after oncogenic signaling blockade by FLT3 inhibitors (FLT3i). Through this integrative approach, we identified activation of innate immune stress response pathways after treatment of FLT3-mutant AML cells with FLT3i. Utilizing genetic approaches, we demonstrated that innate immune pathway activation via IRAK1 and IRAK4 contributes to adaptive resistance in FLT3-mutant AML cells. The immediate nature of IRAK1/4 activation in adaptively resistant FLT3-ITD AML cells requires concomitant inhibition of these targets to avoid compensatory signaling and cell survival. Achieving optimal multi-drug combination regimens that yield extended overlapping exposure while avoiding unwanted toxicities is challenging. Therefore, we desired a small molecule inhibitor that simultaneously targeted the FLT3 and IRAK1/4 kinases to eradicate adaptively resistant FLT3-ITD AML. To overcome this adaptive resistance mechanism, we developed and optimized a novel small molecule that simultaneously inhibits FLT3 and IRAK1/4 kinases. The FLT3-IRAK1/4 inhibitor exhibited potent binding affinity for IRAK1 (KD= 2.9 nM), IRAK4 (KD= 0.3 nM), and FLT3 (KD= 0.3 nM), as well as acceptable pharmacokinetic properties in mice. Moreover, a high-resolution crystal structure demonstrates that the FLT3-IRAK1/4 inhibitor binds as a type I inhibitor (ATP-competitive binding to the active state). The FLT3-IRAK1/4 inhibitor eliminated adaptively resistant FLT3-mutant AML cell lines and patient-derived samples in vitro and in vivo, and displayed superior efficacy as compared to current targeted FLT3 therapies. Our study demonstrates that therapies that simultaneously inhibit FLT3 signaling and compensatory IRAK1/4 activation have the potential to improve the therapeutic efficacy in patients with FLT3-mutant AML. In conclusion, these findings reveal that inflammatory stress response pathways contribute to adaptive resistance in FLT3-mutant AML and suggests that this mechanism may extend to other malignant cells undergoing a stress-induced response to therapy. Disclosures Hoyt: Kurome Therapeutics: Consultancy. Berman:Astellas: Membership on an entity's Board of Directors or advisory committees, Research Funding. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Amgen: Honoraria; Lilly: Honoraria; Gilead: Consultancy; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Roche: Consultancy, Research Funding; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Loxo: Membership on an entity's Board of Directors or advisory committees. Rosenbaum:Kurome Therapeutics: Consultancy, Employment. Perentesis:Kurome Therapeutics: Consultancy. Starczynowski:Kurome Therapeutics: Consultancy.

Description: Inflammatory and innate immune signaling pathways are activated in leukemic stem and progenitor cells and contribute to the pathogenesis of hematologic malignancies, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). UBE2N is a ubiquitin (Ub) conjugating enzyme that catalyzes lysine 63 (K63)-linked Ub chains on substrates that are critical for signal transduction of broad innate immune signaling pathways. Here we report that UBE2N is required for leukemic cell function by mediating oncogenic innate immune signaling, and identified a novel chemical class of small molecule inhibitors that target UBE2N enzymatic activity. Upon UBE2N downregulation with two lentivirally expressed shRNAs, MOLM-13 and THP-1 cells lose their clonogenic potential and undergo cell death. Unlike for AML cells, UBE2N was dispensable for colony formation and viability of healthy cord blood CD34+ cells. The active site of UBE2N contains a cysteine (Cys) at position 87, which is essential for binding and transfer of Ub to its substrates. We performed in silico structure- and in vitro cell-based screens to identify small molecules that dock to the active site of UBE2N and covalently bind to Cys-87, as an approach to inhibit Ub transfer to substrates. Two structurally-related candidates, UC-764864 and UC-764865, emerged as inhibitors of UBE2N, as they specifically blocked the E1-UBE2N thioester transfer in vitro. Treatment of MDS/AML cell lines and patient-derived primary cells with UC-764864 and UC-764865 suppressed innate immune signaling and induced cytotoxic effects in MDS/AML cell lines and primary cells while sparing healthy hematopoietic cells in vitro and in vivo. To identify the molecular basis of UBE2N inhibition, we performed a global Ub screen for changes in ubiquitinated substrates by mass spectrometry and evaluated changes in gene expression by RNA-seq in MOLM-13 cells treated with vehicle control or the newly derived UBE2N inhibitors. RNA-seq of MOLM-13 cells treated with UC-764864 revealed that inhibition of UBE2N in leukemic cells targets oncogenic innate immune pathways, including NF-kB and Type I interferon signaling networks. UC-764864 and UC-764865 reduced the ubiquitination status of UBE2N, and altered the ubiquitination of proteins involved in innate immune signaling and the DNA damage response by primarily reducing K63-linked Ub modifications. Two substrates identified by the Ub screen, DDB1 and UBE2M, are components of the CUL4-CRBN E3 ligase complex and a target of the anti-leukemic therapy, Lenalidomide (LEN). LEN has shown encouraging results in del(5q) MDS patients; however, its effects are limited in other cytogenetic subtypes of MDS or AML. Therefore, the identification of molecular targets that can enhance or extend the use of LEN in a broader spectrum of patients is desired. As such, we explored the possibility of a cooperative effect of LEN and UBE2N inhibitors on MDS/AML cells. As compared to individual treatments, the combination of LEN and UC-764864, UC-764865 or UBE2N shRNAs significantly suppressed the function and viability of MDS/AML cell lines and patient samples in vitro. More striking, treatment of LEN and UBE2N inhibitors impaired MDS/AML cells that are refractory to treatment of LEN or UBE2N inhibitors alone. These findings suggest that UBE2N is a promising target to extend the use of LEN to other subtypes of MDS or AML. In summary, we implicate the Ub conjugating enzyme UBE2N as a target in MDS/AML, and identified novel small molecule inhibitors that target UBE2N and modify the function of Ub E3 ligases that are important for UBE2N-associated diseases, including autoinflammatory and autoimmune disorders, and hematologic malignancies. Disclosures No relevant conflicts of interest to declare.

Description: Myelodysplastic Syndromes (MDS) often progress to Acute Myeloid leukemia (AML) typically following acquisition of mutations in RUNX1, NRAS/KRAS or AXSL1. Inflammatory processes associated with autoimmune diseases or natural aging are thought to contribute to the development of MDS and progression to AML. Moreover, dysregulation of innate immune signaling is broadly observed in hematopoietic stem and progenitor cells (HSPC) from MDS patients. However, how dysregulation of innate immune signaling and/or inflammatory signals might lead to malignant transformation and overt AML has not been fully elucidated. We have developed a mouse model of cell-intrinsic inflammation-driven progression from MDS to AML that will allow us to determine the mechanisms of oncogenic transformation. MicroRNA miR-146a is a 5q gene that is frequently deleted in MDS and AML and its deletion promotes dysregulation of innate immune signaling and a systemic inflammatory state. Deletion of miR-146a results in myeloproliferation and marrow failure in mice associated with attrition of HSPC, but alone is insufficient to induce overt AML. Using a genetically engineered approach, we show that deficiency of miR-146a (miR-146a-/-) combined with a C-terminal truncation of the RUNX1 (S291fsX300) protein causes anemia, thrombocytopenia and ineffective erythropoiesis mimicking human MDS in younger mice, but then the mice progresses to a fatal AML. The inflammatory environment induced by miR-146a deletion was exacerbated in mice with miR-146a-/-expressing RUNX1-S291fsX300 (RUNX1-mut) and correlated with the increased incidence and aggressiveness of hematologic malignancies. In particular, the levels of IFNγ were increased in plasma in mice with miR-146a-/-expressing RUNX1-mut in comparison to mice reconstituted with miR-146a-/-or RUNX1-mut-expressing BM cells. As previously reported, miR-146a-/-cells exhausted during serial transplantation, however RUNX1-mut rescued the hematopoietic repopulation deficiency of miR-146a-/-HSPC, suggesting that the RUNX1 mutation promotes malignant transformation to AML under inflammatory conditions. To establish whether cell-intrinsic immune signaling is required for RUNX1-mut;miR-146a-/- leukemic cells, we evaluated an inhibitor (NSC697923) that targets TRAF6, a key target of miR-146a in MDS/AML, via inhibition of its co-factor UBE2N. Treatment with NSC697923 or a RUNX1 inhibitor (Ro 5-3335) abolished colony formation by RUNX1-mut;miR-146a-/- leukemic cells, indicating that cell-intrinsic innate immune signaling is not only required for initiating AML, but is also critical for sustaining the leukemic phenotype. These findings describe the first evidence that dysregulation of innate immune signaling and/or inflammatory signals contribute to the progression and maintenance of AML. Disclosures Starczynowski: Kurome Therapeutics: Consultancy.

Description: Individuals with clonal hematopoiesis of indeterminant potential (CHIP) are healthy, however they are at an increased risk of developing hematopoietic malignancies. The most frequent mutations in CHIP target DNMT3A and TET2, also are observed in acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), and myelodysplastic syndromes (MDS). These findings indicate that additional alterations are needed for the transition from a pre-leukemic stage to frank leukemia, although the identity of such molecular events remains uncharacterized. To identify cellular states that cooperate with Tet2 loss, we used in vivo RNAi screening and identified the ubiquitin ligase TRAF6 required for malignant transformation of pre-leukemic TET2-deficient hematopoietic stem/progenitor cell (HSPC). Importantly, TRAF6 expression is significantly reduced in 25-50% of AML and MPN patients as compared to healthy controls. Furthermore, TET2 mutations are more strongly correlated with lower expression of TRAF6 as compared to patients with higher TRAF6 expression in certain subsets of AML. To evaluate the consequences of TRAF6 deletion on TET2-deficienct pre-leukemic cells, we generated mice in which TRAF6 and TET2 are conditionally deleted in hematopoietic cells (VavCre;Traf6fl/fl;Tet2fl/fl[DKO]). Traf6KO mice developed a lethal phenotype with signs of MPN, including lymphopenia, neutrophilia, and increased hemoglobin levels; however, this disease was not transplantable. In striking contrast, deletion of TRAF6 in the context of TET2-deficient HSPC resulted in a rapid, penetrant, aggressive, and transplantable MPN/AML. To firmly establish that TRAF6 exhibits tumor suppressor functions, we determined whether physiological levels of TRAF6 overexpression could prevent malignant transformation. Overexpression of TRAF6 in FLT3-ITD mice inhibited malignant myeloid cell expansion in FLT3-ITD mice, and rescued the survival of the animals. To uncover the molecular basis of TRAF6's tumor suppressor function, we performed gene expression profiling and proteomic characterization of TRAF6 ubiquitination substrates in leukemic cells. RNA-sequencing of HSPC revealed that deletion of TRAF6 resulted in a significant overexpression of MYC regulated genes in pre-leukemic HSPC. In support of these findings, the proteomic screen along with extensive in vitro validation experiments identified MYC as a substrate of TRAF6. Unlike the majority of reported ubiquitin-dependent post-translational modifications of MYC, we found that ubiquitination of MYC on Lysine (K) 148 by TRAF6 does not affect its protein stability but rather antagonizes acetylation of MYC on the same lysine and thus suppresses MYC oncogenic activity. We extended these observations to investigate whether inflammatory signaling via Toll-like receptors (TLRs) can antagonize MYC function and suppress leukemic cells. Stimulation of TLRs on leukemic cells resulted in TRAF6-dependent ubiquitination of MYC at K148, which coincided with repositioning of MYC off of its target gene promoters and enhancers, and ultimately in the suppression of leukemic cell viability. Our results demonstrate that TRAF6 functions as a tumor suppressor via its ubiquitination activity that antagonizes K148 acetylation leading to a decrease of MYC transcriptional activity without affecting its protein abundance. Our findings identify TRAF6 as a novel, context-dependent tumor suppressor in myeloid neoplasms, and suggest that innate immune signaling via TLR/TRAF6 could explain why some of the clonal hematopoiesis patients develop AML and others do not. Disclosures Lowe: Blueprint Medicines: Consultancy, Equity Ownership; PMV Pharmaceuticals: Consultancy, Equity Ownership; Petra Pharmaceuticals: Consultancy, Equity Ownership; Constellation Pharma: Consultancy, Equity Ownership; Mirimus: Consultancy, Equity Ownership; ORIC pharmaceuticals: Consultancy, Equity Ownership; Faeth Therapeutics: Consultancy, Equity Ownership. Starczynowski:Kurome Therapeutics: Consultancy.

Description: Myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) disorders in which myeloid cell differentiation is impaired, causing blood lineage cytopenias and potentially leading to acute myeloid leukemia (AML) through malignant transformation. MDS occurs in adults with a median age of 71 years, and is associated with multiple cytogenetic and genetic abnormalities in the diseased HSC. Younger patients can also have MDS as a result of underlying congenital diseases or secondary effects from cancer therapy. It has recently been discovered that some families with high rates of MDS incidence bear heterozygous inherited mutations in DDX41, a member of the DEAD box RNA helicase family of genes. These patients typically have normal hematopoietic indices into adulthood and present with MDS at a median age of 61 years, slightly younger than the general population. Inherited DDX41 mutations are always heterozygous and are typically frame-shift mutations, indicating they are likely loss of function. Approximately half of MDS patients with inherited DDX41 mutations acquire a second-hit, often R525H, in the healthy DDX41 allele in their disease clones. This mutation is also observed in 1-2% of de novo AML patients, suggesting it causes gain of function or dominant negative activity. Multiple functions have been ascribed to DDX41, such as functioning as an innate immune sensor and as an RNA splicing regulator, but its role in the pathogenesis of MDS remains unknown. We set out to model DDX41 mutations by generating conditional DDX41 knockout and R525H-knock-in mice. Combining these alleles and crossing to Rosa-Cre-ERT expressing mice allowed for tamoxifen-inducible acquisition of knockout (KO), heterozygous (HET), heterozygous knock-in (KI/+) and knock-in alone (KI/-) HSPC. The KO and KI/- HSPC were incapable of engrafting into recipient mice and underwent rapid cell cycle arrest and apoptosis, indicating that Ddx41 is required for HSPC cell viability and that the R525H mutation causes loss of the required function. In contrast, the HET and KI/+ HSPC survived and proliferated normally in culture and successfully engrafted irradiated recipient mouse bone marrow. HET and KI/+ transplanted mice had increased numbers of LSK cells, and subset of mice developed a myeloid malignancy, resembling the human disease. To determine the function of DDX41 that is critical for hematopoiesis, we performed a tandem-pulldown followed by mass spectrometry analysis to identify relevant DDX41 interacting proteins in human AML cells. We found that DDX41 interacts with multiple proteins in the small ribosomal subunit, including RPS3 and RPS14. Consistent with disruption of the assembly and function of the small ribosomal subunit, KO and KI/-HSPC exhibited rapid and robust impairment of global protein translation. Polysome profiling indicated an increase in monosomes and a decrease in polysomes in KO cells, consistent with an inability of ribosomes to initiate translation and move along the mRNA. To determine the role of the translation defect in the cell growth deficiency of DDX41-deficient cells, we treated WT, HET, and KO HSPC with the translation inhibitor puromycin and determined that KO cells were relatively more sensitive to translation inhibition, indicating that DDX41-deficient cells are specifically sensitive to further reduction in protein translation. This data supports the conclusion that the cell lethality caused by DDX41 loss is related to ribosome dysfunction. Mechanistically, we find that DDX41-deficient cells have stalled ribosomal RNA (rRNA) processing, characterized by increased unprocessed rRNA and decreased processed rRNA intermediates. In conclusion, we identify a novel function of DDX41 in regulating rRNA processing and ribosome formation that is essential for the survival and proliferation of HSPC. The loss of DDX41 may contribute to MDS as a result of impaired ribosome function, as has been previously reported in patients bearing mutations in other ribosome regulators. Disclosures Starczynowski: Kurome Therapeutics: Consultancy.

Description: Germline mutations in the DEAD-box RNA helicase gene DDX41 cause inherited susceptibility to Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML). These patients have normal hematopoietic indices into adulthood and present with MDS at a median age of 61 years, slightly younger than the general MDS patient population (71 years). Germline DDX41 mutations are always heterozygous and are typically frame-shift mutations, causing loss of function of the protein. More than half of these patients acquire a second-hit mutation in the healthy DDX41 allele in their disease clones. Greater than 80% of the second-hit mutations cause the amino acid substitution R525H, which results in loss of helicase activity. Multiple functions have been ascribed to DDX41, such as functioning as an innate immune sensor, a component of the RNA spliceosome, and a regulator of ribosomal RNA processing; however, its role in the pathogenesis of MDS remains poorly understood. To mimic the DDX41 mutations in human patients, we generated conditional mouse models of the most common DDX41 mutations, D140fs and R525H. We found that DDX41 is essential for the viability and function of hematopoietic stem and progenitor cells (HSPCs) and that R525H mutations render DDX41 inactive. We have also reported that mice with biallelic DDX41 mutations develop progressive anemia, dysplasia, and marrow failure. HSPCs lacking wild-type Ddx41 have dysfunctional ribosomes and reduced protein translation, leading to cycle arrest and apoptosis. As a mechanistic basis for the ribosome defect in Ddx41-deficient HSPCs, we found that loss of Ddx41 causes a profound increase in unprocessed snoRNA transcripts, which likely interrupts their cellular function. Importantly, we did not observe a change in host gene transcript abundance, and thus it is unlikely that the defects observed in Ddx41-deficient cells are caused by loss of the protein products of the host genes. SnoRNAs regulate ribosomal RNA (rRNA) by catalyzing post-transcriptional modifications, including methylation and pseudouridylation. Since the majority of the snoRNAs that were found to be unprocessed in Ddx41-deficient HSPCs were from the SNORA family, which is involved in pseudouridylation, we quantified the abundance of pseudouridine at specific sites in ribosomal RNA and found it to be reduced in Ddx41-deficient HSPCs. Utilizing global high-throughput sequencing approaches to analyze DDX41 binding to RNAs, we found that DDX41 preferentially binds to snoRNAs relative to all other types of RNA in the cells. Thus, DDX41 binds to snoRNAs and is essential for snoRNA processing, snoRNA-mediated rRNA pseudouridylation, and protein translation in HSPCs. These findings provide critical mechanistic insight into the protein translation defect that we and others have observed in DDX41-deficient cells and uncover the basis of ineffective hematopoiesis in MDS patients with DDX41 mutations. Disclosures Starczynowski: Tolero Therapeutics: Research Funding; Kurome Therapeutics: Consultancy, Current equity holder in private company, Research Funding; Captor Therapeutics: Consultancy.