Background: B-acute lymphoblastic leukemia (ALL) is the most common childhood cancer and is driven primarily by oncogenic chromosomal rearrangements and gene fusions. While overall survival rates are nearly 90% in children and about 40% in adults with good- and intermediate-risk ALL subtypes, clinical outcomes of patients with high-risk ALL subtypes, including KMT2A-rearranged (KMT2A-R), are significantly lower, posing an urgent need to develop novel therapeutic strategies for these patient populations. Results: To identify targetable kinases and potential precision medicine approaches in KMT2A-R ALL, we conducted domain-specific kinome-wide CRISPR screens and found the serine/threonine kinase DYRK1A essential for KMT2A-R ALL proliferation. The Cancer Dependency MAP suggests DYRK1A is non-essential for normal tissues, indicating a potential therapeutic window. RT-PCR and Western blot analyses showed increased DYRK1A expression levels in KMT2A-R ALL cell lines and patient-derived xenograft (PDX) samples compared to other leukemia subtypes, suggesting direct regulation by the KMT2A-fusion oncogene. Meta-analysis of ChIP-seq data confirmed direct binding of the KMT2A-fusion protein to the DYRK1A promoter. Inhibition of menin, a tumor suppressor protein that forms a complex with the KMT2A-fusion protein, reduced DYRK1A expression at RNA and protein levels and prevented the binding of the KMT2A-fusion protein to the DYRK1A promoter, validating the direct regulation of DYRK1A by the KMT2A-fusion oncogene. To test the consequence of direct pharmacologic inhibition of DYRK1A using EHT1610 and harmine, we treated multiple KMT2A-R ALL cell lines and PDX models, validating potent inhibition of leukemia cell growth without inducing apoptosis. Furthermore, we detected potent upregulation of MYC and hyperphosphorylation of ERK. Premature ERK signaling activation in developing B cells is known to induce cell cycle arrest as a protective mechanism to prevent the development of autoimmune diseases. We hypothesized that DYRK1A inhibitor-induced ERK hyperactivation leads to cell cycle arrest in KMT2A-R ALL via a similar mechanism. Supporting our hypothesis, combining DYRK1A inhibitors with the MEK inhibitor trametinib rescued KMT2A-R ALL cell proliferation, validating that ERK hyperactivation is the main driver of DYRK1A inhibitor-mediated cell cycle arrest. Interestingly, although DYRK1A inhibition mainly affected cell proliferation, we detected a potent increase in the pro-apoptotic molecule BIM. BIM is inactivated by the anti-apoptotic molecule BCL2, which was also slightly increased after DYRK1A inhibition, indicating that DYRK1A inhibitors could also sensitize KMT2A-R ALL cells to BCL2 inhibition (e.g., venetoclax). In support of our hypothesis, combined treatment with DYRK1A inhibitors and venetoclax synergistically killed KMT2A-R ALL cells in vitro. Given the poor pharmacodynamics and severe toxicity of EHT1610 and harmine, we synthesized a new DYRK1A inhibitor, GNF2133, and tested its activity in vitro against KMT2A-R ALL cell lines and in vivo +/- venetoclax in KMT2A-R ALL PDX models. Importantly, both GNF2133 and venetoclax were well tolerated in xenograft mice, and combination inhibitor therapy resulted in superior inhibition of in vivo leukemia proliferation and long-term animal survival. Conclusion: Our results identify DYRK1A as a critical regulator of ERK signaling in KMT2A-R ALL. DYRK1A inhibition leads to BIM accumulation, sensitizing cells to BCL2 inhibition by venetoclax in vitro and in vivo and resulting in therapeutic benefit in preclinical PDX models that is highly translatable to the clinic.

BackgroundType I interferons (IFN-Is), secreted by hematopoietic cells, drive immune surveillance of solid tumors. However, the mechanisms of suppression of IFN-I-driven immune responses in hematopoietic malignancies including B-cell acute lymphoblastic leukemia (B-ALL) are unknown.MethodsUsing high-dimensional cytometry, we delineate the defects in IFN-I production and IFN-I-driven immune responses in high-grade primary human and mouse B-ALLs. We develop natural killer (NK) cells as therapies to counter the intrinsic suppression of IFN-I production in B-ALL.ResultsWe find that high expression of IFN-I signaling genes predicts favorable clinical outcome in patients with B-ALL, underscoring the importance of the IFN-I pathway in this malignancy. We show that human and mouse B-ALL microenvironments harbor an intrinsic defect in paracrine (plasmacytoid dendritic cell) and/or autocrine (B-cell) IFN-I production and IFN-I-driven immune responses. Reduced IFN-I production is sufficient for suppressing the immune system and promoting leukemia development in mice prone to MYC-driven B-ALL. Among anti-leukemia immune subsets, suppression of IFN-I production most markedly lowers the transcription of IL-15 and reduces NK-cell number and effector maturation in B-ALL microenvironments. Adoptive transfer of healthy NK cells significantly prolongs survival of overt ALL-bearing transgenic mice. Administration of IFN-Is to B-ALL-prone mice reduces leukemia progression and increases the frequencies of total NK and NK-cell effectors in circulation. Ex vivo treatment of malignant and non-malignant immune cells in primary mouse B-ALL microenvironments with IFN-Is fully restores proximal IFN-I signaling and partially restores IL-15 production. In B-ALL patients, the suppression of IL-15 is the most severe in difficult-to-treat subtypes with MYC overexpression. MYC overexpression promotes sensitivity of B-ALL to NK cell-mediated killing. To counter the suppressed IFN-I-induced IL-15 production in MYChighhuman B-ALL, we CRISPRa-engineered a novel human NK-cell line that secretes IL-15. CRISPRa IL-15-secreting human NK cells kill high-grade human B-ALL in vitro and block leukemia progression in vivo more effectively than NK cells that do not produce IL-15.ConclusionWe find that restoration of the intrinsically suppressed IFN-I production in B-ALL underlies the therapeutic efficacy of IL-15-producing NK cells and that such NK cells represent an attractive therapeutic solution for the problem of drugging MYC in high-grade B-ALL.

Prolactin (PRL) is elevated in B-cell-mediated lymphoproliferative diseases and promotes B-cell survival. Whether PRL or PRL receptors drive the evolution of B-cell malignancies is unknown. We measure changes in B cells after knocking down the pro-proliferative, anti-apoptotic long isoform of the PRL receptor (LFPRLR) in vivo in systemic lupus erythematosus (SLE)- and B-cell lymphoma-prone mouse models, and the long plus intermediate isoforms (LF/IFPRLR) in human B-cell malignancies. To knockdown LF/IFPRLRs without suppressing expression of the counteractive short PRLR isoforms (SFPRLRs), we employ splice-modulating DNA oligomers. In SLE-prone mice, LFPRLR knockdown reduces numbers and proliferation of pathogenic B-cell subsets and lowers the risk of B-cell transformation by downregulating expression of activation-induced cytidine deaminase. LFPRLR knockdown in lymphoma-prone mice reduces B-cell numbers and their expression of BCL2 and TCL1. In overt human B-cell malignancies, LF/IFPRLR knockdown reduces B-cell viability and their MYC and BCL2 expression. Unlike normal B cells, human B-cell malignancies secrete autocrine PRL and often express no SFPRLRs. Neutralization of secreted PRL reduces the viability of B-cell malignancies. Knockdown of LF/IFPRLR reduces the growth of human B-cell malignancies in vitro and in vivo. Thus, LF/IFPRLR knockdown is a highly specific approach to block the evolution of B-cell neoplasms.

Background: KMT2A-rearranged (R) ALL is a high-risk disease with a frequency of 75% in infants and 10% in children and adults with ALL and is associated with chemoresistance, relapse, and poor survival. 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 that DYRK1A is specifically required for the survival of KMT2A-R ALL cell. DYRK1A is a member of the dual-specificity tyrosine phosphorylation-regulated kinase family and has been reported as negatively regulator of cell proliferation. Results: We performed a kinome-wide CRISPR screen in human ALL cell lines and PDX models and identified DYRK1A as a novel target in KMT2A-R ALL. DYRK1A is a serine-threonine kinase with a proposed, but poorly defined role in cell cycle regulation. We performed a meta-analysis of multiple ChIP-Seq experiments and identified that oncogenic KMT2A fusions directly bind to the DYRK1A promoter. Our RT-PCR and Western blot analyses of KMT2A-R ALL cells treated with a menin inhibitor (MI-503) to disrupt the transcriptional activity of the KMT2A-R complex resulted in the downregulation of DYRK1A, indicating that DYRK1A is directly regulated by the KMT2A fusion complex. We further observed that pharmacologic inhibition of DYRK1A with EHT1610 induced potent leukemic cell growth inhibition in vitro and in vivo, demonstrating that DYRK1A could be a new therapeutic target in KMT2A-R ALL cells. To further elucidate the mechanism of DYRK1A function, we treated several KMT2A-R ALL cell lines in vitro with EHT1610, which surprisingly resulted in the upregulation of MYC and hyperphosphorylation of the RAS/MAPK target ERK. Given that ERK hyperactivation stops B cell proliferation during early B cell development to allow them to rearrange their B cell receptor, we hypothesized that cell cycle inhibition upon ERK hyperactivation remains as a conserved mechanism of cell cycle regulation in KMT2A-R ALL. Strikingly, combining DYRK1A inhibition with the MEK inhibitor trametinib antagonistically rescued KMT2A-R ALL cell proliferation, indicating that ERK hyperactivation is the main driver of DYRK1A inhibitor mediated cell cycle arrest. Given that DYRK1A inhibitor does not induce apoptosis and cells restart cell proliferation after EHT1610 withdrawal we concluded that a DYRK1A monotherapy may not be an ideal new treatment option. However, it has been reported that increased MYC activity induces the accumulation of BIM in Burkitt's Lymphoma. Given the increased expression of MYC following DYRK1A inhibition we performed a new Western blot analysis and validated increased expression of BIM in our KMT2A-R ALL cell lines after EHT1610 treatment. To test if targeting the interaction of BIM with BCL2 will induce an apoptotic effect when combined with EHT1610, we treated four KMT2A-R ALL cell lines with increasing concentrations of EHT1610 and the BCL2 inhibitor venetoclax. Strikingly, the combination of DYRK1A inhibition with BCL2 inhibition synergistically killed KMT2A-R ALL cells. Conclusion: Our results validate DYRK1A as an important molecule to regulate cell proliferation via inhibition of MYC and ERK. Targeting DYRK1A results in the accumulation of BIM, which renders the cells sensitive to BCL2 inhibition via venetoclax. While further in vivo studies are needed, we predict that combining DYRK1A inhibition with venetoclax may be a novel precision medicine strategy for the treatment of KMT2A-R ALL. Figure 1 Figure 1. Crispino: Forma Therapeutics: Research Funding; Scholar Rock: Research Funding; MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy. Tasian: Aleta Biotherapeutics: Consultancy; Gilead Sciences: Research Funding; Kura Oncology: Consultancy; Incyte Corporation: Research Funding. Carroll: Incyte Pharmaceuticals: Research Funding; Janssen Pharmaceutical: Consultancy.

KMT2A -rearranged ( KMT2A -R) B cell acute lymphoblastic leukemia (ALL) is a high-risk disease in children and adults that is often chemotherapy resistant. To identify non-cytotoxic approaches to therapy, we performed a domain-specific kinome-wide CRISPR screen in KMT2A -R cell lines and patient derived xenograft samples (PDX) and identified dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) as a potential target. Pharmacologic inhibition of the KMT2A-fusion transcriptional co-regulator Menin released the KMT2A-fusion complex from the DYRK1A promoter thereby lowering DYRK1A expression levels confirming DYRK1A as a direct target of the KMT2A fusion oncogene. Direct pharmacologic inhibition of DYRK1A decreased cell proliferation of KMT2A -R ALL, thereby confirming the requirement of DYRK1A in this ALL subtype. To further understand the biologic function of DYRK1A in KMT2A -R ALL, we leveraged pharmacologic DYRK1A inhibitors in KMT2A -R PDX and cell line models. DYRK1A inhibition consistently led to upregulation of MYC protein levels, and hyperphosphorylation of ERK, which we confirmed via in vivo treatment experiments. Furthermore, DYRK1A inhibition decreased ALL burden in mice. Our results further demonstrate that DYRK1A inhibition induces the proapoptotic factor BIM, but ERK hyperphosphorylation is the driving event that induces cell cycle arrest. In contrast, combined treatment of KMT2A -R ALL cells in vitro and in vivo with DYRK1A inhibitors and the BCL2 inhibitor, venetoclax, synergistically decreases cell survival and reduced the leukemic burden in mice. Taken together these results demonstrate a unique function of DYRK1A specially in KMT2A -R ALL. Synergistic inhibition of DRYK1A and BCL2 may provide a low-toxic approach to treat this high risk ALL subtype. ### Competing Interest Statement Dr. Crispino is on the SAB for Alethiomics and consults for Cellarity. SKT has served on scientific advisory boards for Kura Oncology and Syndax Pharmaceuticals and receives research funding from Kura Oncology for pediatric studies of menin inhibition.

Cytotoxic natural killer cells kill tumors and infected cells. We carried out CRISPR-based gene editing and transcriptional regulation in hard-to-manipulate NK-92 cells. NK-92-based therapies were found to be safe and efficacious in preclinical studies of cancers. Here, we have pioneered the generation and validation of NK-92 cells constitutively expressing Cas9 or dCas9 for knockout (CRISPRko), transcriptional activation (CRISPRa), or transcriptional repression (CRISPRi) of genes. Our CRISPR-engineered NK-92 cell platforms can be modified for research and off-the-shelf therapeutic applications.

Abstract Rationale B cell malignancies, including leukemia and lymphoma, are high-risk lymphoid neoplasms. B cell malignancies predispose to autoimmune diseases including systemic lupus erythematosus (SLE) which increase the risk of developing these malignancies by >5-fold. Increased prolactin (PRL) expression is known to exacerbate SLE and promote the survival of autoreactive B cells. Furthermore, PRL induces expression of the protooncogenes, MYC and BCL2, in lymphoid tissues. However, whether PRL drives the initiation and maintenance of B cell malignancies was not known. Results We first tested our hypothesis that PRL, specifically signaling through the pro-proliferative and anti-apoptotic long isoform (LF) of the PRL receptor (PRLR), drives the progression of SLE to B cell malignancies. To this end, we knocked down the LF PRLR in MRL-lpr mice predisposed to developing SLE using a splice-modulating oligomer (SMO) that blocks splicing to produce the LF PRLR without affecting the short isoforms. LF PRLR knockdown reduced splenic and circulating B cell numbers in MRL-lpr SLE mice (Fig.1a). Consistent with reduced B cell numbers, BCL2 expression in B cells of SLE mice was suppressed after LF PRLR knockdown, although MYC was unaltered (Fig.1b). By sequencing the immunoglobulin heavy chains (IGH), we compared the composition of the splenic B cell repertoire between control- and LF PRLR SMO-treated SLE mice. Control oligomer treated SLE mice accumulated splenic B cells with long complementary determining region 3 (CDR3) and B cells with non-functional IGH, characteristics of autoreactive B cells. Treatment with the LF PRLR SMO reduced both. We then measured the expression of enzymes known to induce malignant transformation of B cells, namely recombination activating genes 1/2 (RAG1/2) and activation-induced cytidine deaminase (AID), in B cells of SLE mice in controls versus LF PRLR knockdown. Importantly, LF PRLR knockdown significantly reduced RAG1 (Fig.1c) and AID expression in splenic B cells of SLE mice (Fig.1d,e). Our findings thus underscore a causal role for LF PRLR signaling in promoting of malignant transformation of B cells in SLE. Because PRL induces the expression of BCL2 and MYC in lymphocytes, we next determined whether LF PRLR promotes the survival of overt B cell malignancies that overexpress MYC and BCL2, including diffuse large B cell lymphoma (DLBCL) and B-cell acute lymphoblastic leukemia (B-ALL). We observed that B-lymphoblasts expressed significantly higher levels of PRL and the LF PRLR as compared to normal B cells (Fig.1f). We also found that higher expression of PRL at diagnosis predicts poor clinical outcome in DLBCL patients (P=0.0244), and that patients with MYC/BCL2-overexpressing ALLs with a poor prognosis had significantly higher expression of the LF PRLR compared to their MYC lowBCL2 low counterparts (P<0.0001). These observations suggested that LF PRLR may modulate MYC and BCL2 expression. Knockdown of the LF PRLR using the LF PRLR SMO in MYC/BCL2-driven human B cell malignancies killed lymphoblasts and reduced MYC and BCL2 protein levels (Fig.1g). Because we previously showed that MYC-driven lymphoid malignancies are sensitive to natural killer (NK) cell-mediated immune clearance, we also examined whether LF PRLR knockdown synergized with NK cells in killing DLBCL. We found that LF PRLR knockdown enhanced NK cell-mediated killing of B-lymphoblasts (Fig.1h). Of note, no reductions were observed in NK cell viability or MYC levels within NK cells upon LF PRLR knockdown, suggesting that LF PRLR selectively kills B-lymphoblasts without negatively impacting NK homeostasis. Conclusion Our studies identify the specific knockdown of LF PRLR as a potentially safe and targeted strategy to prevent the onset of B cell malignancies in SLE patients and to treat flagrant DLBCL and B-ALL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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.