Bending in plant tissues results from differential cell elongation. We have characterized Arabidopsis "hookless" mutants that are defective in differential growth in the hypocotyl. HOOKLESS1 was cloned and its predicted protein shows similarity to a diverse group of N-acetyltransferases. HOOKLESS1 mRNA is increased by treatment with ethylene and decreased in the ethylene-insensitive mutant ein2. High level expression of HOOKLESS1 mRNA results in constitutive hook curvature. The morphology of the hookless hypocotyl is phenocopied by inhibitors of auxin transport or by high levels of endogenous or exogenous auxin. Spatial patterns of expression of two immediate early auxin-responsive genes are altered in hookless1 mutants, suggesting that the ethylene response gene HOOKLESS1 controls differential cell growth by regulating auxin activity.

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Recurrent internal tandem duplication (ITD) mutations are observed in various cancers including acute myeloid leukemia (AML), where ITD mutations in tyrosine kinase receptor FLT3 are associated with poor prognostic outcomes. Several FLT3 inhibitors (FLT3i) are in clinical trials for high-risk -ITD-positive AML. However, the variability of survival following FLT3i treatment suggests that the mere presence of-ITD mutations might not guarantee effective clinical response. Motivated by the heterogeneity of -ITD mutations, we investigated the effects of-ITD structural features on the response of AML patients to treatment. We developed the HeatITup (HEAT diffusion for Internal Tandem dUPlication) algorithm to identify and quantitate ITD structural features including nucleotide composition. Using HeatITup, we studied the impact of ITD structural features on the clinical response to FLT3i and induction chemotherapy in -ITD-positive AML patients. HeatITup accurately identifies and classifies ITDs into newly defined categories of "typical" or "atypical" based on their nucleotide composition. A typical ITD's insert sequence completely matches the wild-type , whereas an atypical ITD's insert contains nucleotides exogenous to the wild-type Our analysis shows marked divergence between typical and atypical ITD mutation features. Furthermore, our data suggest that AML patients carrying typical ITDs benefited significantly more from both FLT3i and induction chemotherapy treatments than patients with atypicalITDs. These results underscore the importance of structural discernment of complex somatic mutations such as ITDs in progressing toward personalized treatment of AML patients, and enable researchers and clinicians to unravel ITD complexity using the provided software.

Background: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype occurring in 15-40% of older children, adolescents, and adults with B-ALL and is associated with high relapse rates and poor survival. Approximately 50% of Ph-like ALL cases have CRLF2 (cytokine receptor-like factor 2 rearrangements (CRLF2-R), often with concomitant JAK2 point mutations. We and others have shown that targeted inhibition of JAK/STAT and PI3K/AKT signaling in CRLF2-rearranged Ph-like ALL only partially inhibits leukemia proliferation. Our preliminary data indicates that modified B cell receptor signaling results in ERK activation and Ph-like ALL cell growth that appears independent of CRLF2-activated signaling. This study aimed to define the extent to which BCR signaling mediates drug resistance in Ph-like ALL after cytokine receptor signaling inhibition and to develop novel combinatorial treatment strategies. Methods and Results: We first validated high basal levels of JAK/STAT and PI3K pathway phosphoproteins via immunoblotting of cell lysates from >20 primary childhood and adult Ph-like ALL samples and patient-derived xenograft (PDX) models. Despite lack of B-cell receptor (BCR) μ heavy chain on Ph-like ALL cells by flow cytometry analysis, we detected high expression levels of the BCR-associated proteins CD79A, CD79B, and BLNK. We further identified constitutive phosphorylation of downstream BCR-associated signaling molecules, including SRC family kinases (SFKs), BTK, and ERK. Given these new observations, we then sought to delineate more precisely alternative pathways in Ph-like ALL (beyond JAK/STAT and PI3K) that may contribute to adaptive signaling regulation and confer resistance to kinase inhibitor monotherapy. We hypothesized that JAK inhibitor (JAKi) resistance is mediated by compensatory activation of PI3K/AKT/mTOR through 'BCR-like' signaling via CD79/CD19/SRC/BTK/ERK. To test this hypothesis, we treated CRLF2-R Ph-like ALL cells in vitro with selective inhibitors of JAK1/2 (ruxolitinib) or PI3Kd (INCB050465) and assessed effects upon signaling via immunoblotting. We observed i) effective dephosphorylation of STAT5 and moderate dephosphorylation of AKT with full reactivation within 72h following JAKi or PI3Kdi monotherapy, respectively, ii) combined JAKi and PI3Kdi prevented PI3K pathway, reactivation, and iii) JAKi and PI3Kdi had no effects upon ERK signaling. These results suggested that oncogene-independent activation of BCR and PI3K pathway signaling occurs in Ph-like ALL. We then performed immunofluorescence analysis and detected distinct clustering and homodimerization of CD79B at the cell membrane of Ph-like ALL cells, as well as physical proximity of CD79B and CD19, suggesting that CD19 could be phosphorylated by SRC/SFKs independently of an activated BCR. Interestingly however, CRISPR-mediated deletion of CD19 or CD79B resulted in hyperactivation of ERK signaling. Finally, we tested if triple inhibition of JAK/STAT, PI3K, and SRC/SFKs signaling is sufficient to eradicate Ph-like ALL. Strikingly, combined JAKi (ruxolitinib), PI3Kdi (INCB050465), and SRCi (dasatinib) silenced STAT5, PI3K, and ERK signaling and induced near-complete cytotoxicity in vitro and in vivo in Ph-like ALL cell lines and patient-derived xenograft (PDX) models. While triple inhibitor treatment appeared well-tolerated in mice, we also assessed dual kinase inhibition of JAK2 and PI3K with dexamethasone (a known repressor of BCR signaling) as a more clinically relevant combinatorial strategy. We observed similarly robust inhibition of signaling pathways and leukemia eradication in vitro and in vivo in our Ph-like ALL cell lines and PDX models. Conclusions: Children and adults with Ph-like ALL have a >60% risk of relapse despite maximally intensive chemotherapy, demonstrating need for alternative therapeutic approaches. While earlier studies by our group and others have reported partial anti-leukemia activity of JAKi and PI3Ki monotherapies, our new data provide compelling evidence that three essential cooperating signaling pathways are required for Ph-like leukemogenesis. We postulate that triple JAK, PI3K, and SRC inhibition or dual kinase inhibition with chemotherapy can overcome innate compensatory mechanisms of signaling resistance and may in fact be necessary to eradicate Ph-like ALL in patients. Disclosures Perl: Daiichi Sankyo: Consultancy, Honoraria, Other, Research Funding; Arog: Consultancy, Other: Non-financial support included travel costs for advisory board meetings.; AbbVie: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; Actinium Pharmaceuticals: Consultancy, Honoraria, Other: Clinical Advisory Board member, Research Funding; Agios: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Non-financial support included travel costs for advisory board meetings.; Jazz: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; NewLink Genetics: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; Astellas: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings as well as a medical writing company that assisted with manuscript preparation/submission and slide deck assembly for academic meeting presentations of trial data., Research Funding; Takeda: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; Bayer: Research Funding; BioMed Valley Discoveries: Research Funding; FujiFilm: Research Funding; Novartis: Honoraria, Other: Advisory board, Non-financial support included travel costs for advisory board meetings as well as a medical writing company that assisted with manuscript preparation/submission and slide deck assembly for academic meeting presentations of the data., Research Funding. Carroll:Astellas Pharmaceuticals: Research Funding; Incyte: Research Funding; Janssen Pharmaceuticals: Consultancy. Tasian:Gilead Sciences: Research Funding; Incyte Corportation: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees.

Background: KMT2A-rearranged (R) ALL is associated with chemoresistance, relapse, and poor survival with a frequency of 75% in infants and 10% in children and adults with ALL. Current intensive multiagent chemotherapy regimens induce significant side effects, yet fail to cure many patients, demonstrating continued need for novel therapeutic approaches. We performed a kinome-wide CRISPR screen and identified DYRK1A as required for KMT2A-R ALL cell survival, but not in other high risk ALL genetic subtypes. DYRK1A is a member of the dual-specificity tyrosine phosphorylation-regulated kinase family and has been reported as a critical oncoprotein in a murine Down syndrome model of megakaryoblastic leukemia. DYRK1A negatively regulates cell proliferation and induces quiescence. Paradoxically, genetic deletion or pharmacological inhibition of DYRK1A upregulates the cell cycle regulator CCND3 and increased numbers of B cells in S-phase, yet also significantly reduces cell proliferation. The specific role of DYRK1A in ALL has not been reported. Results: We assessed the importance of DYRK1A deletion in a focused screen of 14 previously identified kinases. Meta-analysis of ChIP-Seq data from two KMT2A-AFF1 cell lines and a human KMT2A-Aff1-FLAG transduced ALL model demonstrated direct binding of both N-terminal (KMT2AN) and C-terminal (AFF1C) and the FLAG-tagged KMT2A-fusion to the DYRK1A promoter. To assess if KMT2A fusion directly regulates DYRK1A expression, we treated SEM cells with the menin-KMT2A disrupter MI-503 and identified that the KMT2A fusion protein is a positive regulator of DYRK1A. Pharmacologic inhibition of DYRK1A with EHT1610 demonstrated potent leukemic cell growth inhibition, demonstrating that DYRK1 could be a new therapeutic target in KMT2A-R ALL. To further elucidate the mechanism of DYRK1A function, we treated several KMT2A-R ALL cell lines in vitro with EHT1610, which resulted in accumulation of CCND3 as expected. In addition, we detected upregulation of the positive cell cycle regulator MYC and the replication stress response molecule CHK1. In a second experiment, we validated the upregulation of MYC and identified significant upregulation of the proapoptotic protein BIM. Strikingly, meta-analysis of gene expression data from Dyrk1a-deleted murine pre-B cells isolated from a conditional Dyrk1a knockout mouse model also demonstrated increased levels of MYC and CHK1, validating that the EHT1610 mediated upregulation of MYC or CHK1 is a specific effect induced by DYRK1A inhibition. Western blot analysis demonstrated that KMT2A-R ALL cell lines have constitutive activation of pH2AX. Based on these data, we hypothesize that DYRK1A-mediated upregulation of CCND3 and MYC forces the cells to proliferate, which significantly increases replication stress and causes apoptosis, as evident by upregulation of CHK1 and BIM. To test if targeting the interaction of BIM with BCL2 will have an increased apoptotic effect when combined with EHT1610, we treated two KMT2A-R ALL cell lines with increasing concentrations of EHT1610 and the BCL2 inhibitor venetoclax. Strikingly, we observed a synergistic effect with both drugs, suggesting that combining these inhibitors has superior anti-leukemic activity. Conclusions: DYRK1A and MYC are positively regulated by the KMT2A fusion protein in KMT2A-R ALL and negatively regulate each other. Pharmacologic inhibition of DYRK1A resulted in significant growth disadvantage of KMT2A-R ALL cells due to increased MYC and CHK1 proteins that induce replication stress. While further in vivo studies are needed, we predict that combining DYRK1A inhibition with venetoclax may be a novel precision medicine strategy for KMT2A-R ALL that is translatable to the clinic for patients with these high-risk leukemias. Disclosures Tasian: Gilead Sciences: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Research Funding.