ALBERT

Description: We previously demonstrated that CRM1, a major nuclear export factor, accumulates at Hox cluster regions to recruit nucleoporin-fusion protein Nup98HoxA9, resulting in robust activation of Hox genes (Oka et al., 2016). However, whether this phenomenon is general to other leukemogenic proteins remains unknown. Here, we show that two other leukemogenic proteins, nucleoporin-fusion SET-Nup214 and the NPM1 mutant, NPM1c, which contains a nuclear export signal (NES) at its C-terminus and is one of the most frequent mutations in acute myeloid leukemia, are recruited to the HOX cluster region via chromatin-bound CRM1, leading to HOX gene activation in human leukemia cells. Furthermore, we demonstrate that this mechanism is highly sensitive to a CRM1 inhibitor in leukemia cell line. Together, these findings indicate that CRM1 acts as a key molecule that connects leukemogenic proteins to aberrant HOX gene regulation either via nucleoporin-CRM1 interaction (for SET-Nup214) or NES-CRM1 interaction (for NPM1c).

Description: Ph-like acute lymphoblastic leukemia (also known as BCR-ABL1-like ALL) is a new disease entity of B-cell ALL (B-ALL) that exhibits a mRNA expression profile similar to that of Philadelphia chromosome-positive ALL (Ph+ ALL). Ph-like signature is presumably driven by kinase-activating gene alterations. Thus, both gene expression pattern and DNA mutational status should be assessed to make a definitive diagnosis for Ph-like ALL. A variety of approaches combining multiple methods, including RNA sequencing (RNA-seq), Taqman low-density array (LDA), fluorescence in situ hybridization (FISH) and targeted DNA sequencing, are being tested; however, such multi-omics approaches are available only in limited institutions. Since Ph-like ALL patients generally exhibit poor response to standard chemotherapy, and tyrosine kinase inhibitors (TKIs) may benefit them when used in a timely manner, a fast, accurate and generalizable diagnostic method is critically needed. In the present study, we have developed a nCounter-based diagnostic method for Ph-like ALL and validated it using a cohort of Japanese adult B-ALL cases. To identify genes that are uniquely expressed (or not expressed) in Ph+ B-ALL, we first obtained publicly-available gene expression datasets comprising 1146 B-ALL cases and identified 82 differentially-expressed genes in Ph+ ALL cases. We then assessed expression levels of those genes in an independent cohort using the nCounter, which enables fast, sensitive and accurate RNA detection. We also tested whether nCounter-based methods can detect fusion transcripts relevant for Ph-like ALL pathogenesis using probes targeting ABL1, ABL2, CSF1R, PDGFRB, and JAK2. We analyzed 123 samples (Ph+ = 42, Ph- = 81, age 16 to 67) obtained from newly-diagnosed adult B-ALL patients enrolled in two clinical trials conducted by the Fukuoka Blood and Marrow Transplantation Group (FBMTG) (Nagafuji et al. Eur J Haematol 2019). Unsupervised hierarchical clustering successfully stratified 123 cases into two disease clusters: Ph+ and Ph- subgroups. As expected, Ph+ subgroup included almost all Ph+ ALL cases (40 out of 42 cases), while 18 out of 81 Ph- ALL cases (22%) were categorized into the Ph+ subgroup. We defined these cases as Ph-like ALL. To validate the nCounter-based Ph-like ALL classification, we performed RNA-seq and target-capture DNA sequencing of all Ph- ALL cases. As expected, we detected kinase-activating fusions/rearrangements, including CRLF2 rearrangements (7 cases), PDGFRB fusions (3 cases), JAK2 fusions (2 cases), EPOR rearrangements (2 cases), ABL1 fusion (1 case), and FLT3 internal tandem duplication (1 case) in 16 Ph-like ALL cases, while no genetic alterations were detected in 2 cases. Fusion genes involving PDGFRB were consistently detected by nCounter (3/3); however, detection of those involving JAK2 (1/2) and ABL1 (0/1) were inconsistent. JAK2 and/or RAS mutations were detected in 5 of 7 Ph-like ALL cases harboring CRLF2 rearrangements. Of note, CRLF2 protein expression was detected by FACS in all CRLF2-rearranged cases. We next assessed significance of the Ph-like signature on clinical outcomes using a cohort of 40 Ph- ALL cases, in which minimal/measurable residual disease (MRD) status, assessed by IgH and/or TCR rearrangements, as well as clinical data were available (Nagafuji et al. Eur J Haematol 2019). Ph-like ALL cases exclusively exhibited MRD positivity after induction therapy as compared to non-Ph-like cases (p=0.04), indicative of the chemo-resistant nature of Ph-like ALL as previously reported (Roberts et al. N Engl J Med, 2014 and Roberts et al. J Clin Oncol, 2017). As expected, Ph-like ALL cases exhibited significantly poor disease-free survival compared with non-Ph-like ALL cases (p=0.04); however, no significant difference was evident in overall survival (p=0.62) presumably due to the fact that all MRD-positive cases were subjected to allo-HSCT after induction therapy. These data indicate that MRD-based therapy stratification could overcome chemo-resistant nature of Ph-like ALL. Our data suggest that nCounter-based diagnostic method is fast and accurate to identify Ph-like ALL. Since Ph-like signature generally dictates poor clinical outcomes, and upfront TKI therapy may improve them, our method could facilitate precision medicine in the treatment of Ph- B-ALL. Disclosures Akashi: Sumitomo Dainippon, Kyowa Kirin: Consultancy; Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding.

Description: Progress has been made in deciphering molecular mechanisms underlying AML pathogenesis due in part to near-complete understanding of AML genomes. However, AML is yet a devastating disease with a long-term survival rate of less than 30%, underscoring an urgent need for the development of additional therapeutic modalities. To identify novel targets for AML therapy, we performed genome-wide CRISPR-Cas9 dropout screens employing two mouse AML cell lines (CALM/AF10 and MLL/AF9), followed by a second screen in vivo. These two cell lines, which we established, harbor wild-type (WT) Trp53with normal karyotype, enabling us to interpret screening results more easily due to a "clean" genetic background. We then validated our findings using human AML cell lines and patient-derived xenograft (PDX) models (Yamauchi et al. Cancer Cell 2018). In the current study, we assessed the screening results furtherusing MAGeCK MLE program (Li et al. Genome Biology 2015)and the DepMap (https://depmap.org/), a publicly available genome-wide CRISPR-Cas9 screen datasets of cancer cell lines including 15 human AML cell lines. We show that PAICS (Phosphoribosylaminoimidazole carboxylase), which encodes an enzyme involved in de novo purine biosynthesis, is a molecule essential for AML cell survival. MRT252040, a newly-developed PAICS inhibitor (PAICSi), efficiently killed AML cell lines with different genetic backgrounds and significantly prolonged survival of AML PDX models. Furthermore, we investigated the mechanism action of PAICSi employing additional functional screens: CRISPR-Cas9 mutagenesis scan of all Paicscoding exons and a genome-wide CRISPR/Cas9 dropout screen in the presence of PAICSi. Read counts for each Paics-targeted single-guide RNA (sgRNA) significantly decreased in vitro (AML cell lines) and in vivo (mouse AML model). We then assessed the functional significance of PAICS inhibition in AML cell survival via shRNA-mediated PAICSknockdown. AML cells expressing PAICS shRNA exhibited a proliferative disadvantage compared to non-transduced cells or those expressing scrambled shRNA, indicating a toxic effect of PAICS depletion in AML cells. We next asked whether inhibition of PAICS enzymatic activity affects AML cell proliferation and/or apoptosis using PAICSi. We assessed AML growth rate, cell cycle status and apoptosis and found that inhibition of PAICS enzymatic activity delays AML cell proliferation via inducing cell cycle arrest and apoptosis. As expected, CRISPR-Cas9 mutagenesis scan showed that sgRNAs targeting the exonic regions relevant to PAICS enzymatic activity were significantly decreased after the 16-day incubation. We next performed genome-wide CRISPR-Cas9 screens in the presence of PAICSi, followed by second screens using a small-scale sgRNA library containing 8-10 sgRNAs per candidate gene.We identified genes potentially involved in PAICSi resistance as well as those whose loss are synthetic lethal to PAICS inhibition. X-box-binding protein 1 (Xbp1) was among the top hits in the genes relevant to PAICSi resistance genes, and sgRNAs targeting Xbp1significantly enriched in the presence of PAICSi. In contrast, sgRNAs targeting Slc43a3or Hprt, both of which are implicated in the purine salvage pathway, were significantly dropped-out, indicating that PAICSi-mediated anti-leukemia effects can be enhanced upon concurrentinhibition of the purine salvage pathway. Finally, we explored potential anti-leukemia effects of PAICSi in vivo using AML PDX models established from two human AML lines. PAICSi exhibited anti-leukemic activity, as evidenced by the lower leukemia burden in peripheral blood and bone marrow of PAICSi-treated mice. They survived significantly longer than vehicle-treated mice, indicative of therapeutic efficacy of PAICSimonotherapy against AML in vivo. In summary, we identified PAICS as an essential gene for AML cell survival. We propose that pharmacological targeting of the de-novo purine synthesis pathway via PAICSi is a potential therapeutic strategy for AML therapy. Disclosures Akashi: Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding; Sumitomo Dainippon, Kyowa Kirin: Consultancy.

Description: The BCL-2 protein family plays a key role in leukemogenesis via counteracting proapoptotic signals, thereby enhancing leukemia cell survival. Venetoclax, a selective BCL-2 inhibitor, has been proven effective against AML in combination with hypomethylating agents. Since MCL1, another anti-apoptotic protein, is reportedly more abundant than BCL-2 in AML cells and associated with resistance to chemotherapy, development of clinical-grade MCL1 inhibitors has been highly regarded. In fact, a series of MCL1 inhibitors are currently being tested in clinical trials. While relationships between metabolic condition in mitochondria and sensitivity to Venetoclax have been proposed in recent studies, molecular mechanisms underlying MCL1 inhibitor resistance are not well understood. To identify genes/pathways whose loss induce MCL1 inhibitor resistance in AML cells, we performed genome-wide CRISPR-Cas9 screens using a mouse AML cell line in the presence or absence of s63845, a MCL1 inhibitor (Kotschy et al. Nature 2016). To establish AML cell lines with a relatively clean genetic background, we first established AML in mice by transducing the MLL/AF9 leukemia oncogene into mouse bone marrow stem/progenitor cells ex vivo, followed by serial transplants (Yamauchi et al. Cancer Cell 2018). We then harvested AML cells from leukemic mice and established AML cell lines with normal karyotype and intact Trp53 activity. We then performed genome-wide CRISPR-Cas9 screens in the presence or absence of s63845, followed by a second screen with a small-scale library targeting genes that were negatively- or positively-selected upon s63845 treatment in the primary screen. Genes relevant for S63845 resistance were identified using MAGeK MLE (Li et al. Genome Biology 2015) and DrugZ (Colic et al. BioRxiv 2019) programs. Single-guide RNAs (sgRNAs) targeting intrinsic pro-apoptotic genes, such as Bak1, Casp9 and Apaf1, were enriched exclusively in the presence of s63845, attesting to the validity of our experiment. To identify genes relevant to s63845 resistance, we focused on the genes whose sgRNAs were enriched exclusively upon s63845 treatment. We found that loss of Me2, which encodes a mitochondrial malic enzyme that catalyzes the oxidative decarboxylation of malate to pyruvate, promotes AML cell survival only in the presence of s63845, but not in vehicle- or Venetoclax-treated cells. This finding was validated in the second screen, in which 8 independent sgRNAs targeting Me2 were tested. We next generated two independent Me2-null mouse AML cell lines (MLL/AF9 and CALM/AF10) using sgRNAs targeting Me2. Me2-null cells exhibited survival advantage over control cells upon s63845 treatment, revealed by cell proliferation assay. To determine at which step of the apoptotic pathway Me2 deficiency exerts s63845 resistance, we assessed MOMP (mitochondrial outer membrane permeabilization) levels by FACS with or without s63845 treatment. Me2-null cells released less cytochrome C than Me2-wild type (WT) cells upon s63845 treatment. As expected, Me2-null cells exhibited less Annexin-positivity than WT cells upon MCL1 inhibition. Importantly, both mRNA and protein levels of BCL-2 family members, including Bcl-2, Mcl1, Bcl2L1, Bax and Bak1, were comparable regardless of Me2 status. We next performed CRISPR-Cas9 saturation mutagenesis scan of all ME2 exons in the presence or absence of s63845 using Molm-13, a human AML cell line. sgRNAs targeting ME2 coding exons were enriched only in the presence of s63845, while those targeting ME2 3'UTR were unchanged/neutral regardless of experimental conditions. In conclusion, using an unbiased, genome-wide CRISPR/Cas9 screens, we identified Me2, a mitochondrial metabolic enzyme, as a factor relevant for MCL1 inhibitor resistance. Our study may facilitate the understanding of molecular mechanisms underlying acquired resistance to MCL1 inhibitors in AML. Disclosures Akashi: Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding; Sumitomo Dainippon, Kyowa Kirin: Consultancy.

Description: Acute myeloid leukemia (AML) is a devastating disease with a long-term survival rate of less than 30%. While AML is a genetically heterogeneous disease, TP53 mutation is among the most powerful risk factors in AML, underscoring the critical need to devise a novel therapeutic strategy for TP53-mutated AML. To identify genes/pathways whose loss are vulnerable to TP53 deficiency in AML cells, we performed genome-wide CRISPR-Cas9 screens using Trp53-knockout (KO) and wild-type (WT) mouse AML cells. To generate AML lines with a relatively "clean" genetic background, we established mouse AML lines harboring WT Trp53 with normal karyotype (Yamauchi et al. Cancer Cell 2018). We then generated Trp53-KO AML lines using a single guide RNA (sgRNA) targeting Trp53. Genome-wide CRISPR-Cas9 dropout screens were performed using these lines to identify genes/pathways whose loss are vulnerable to TP53 deficiency. Importantly, sgRNAs targeting Trp53 were enriched only in Trp53-WT AML cells, but not in Trp53-KO cells. Furthermore, sgRNAs targeting Mdm2, which encodes a E3 ubiquitin-protein ligase for Trp53 protein, were depleted only in Trp53-WT cells, attesting to the validity of our experimental system. We identified Xpo7, a putative nuclear/cytoplasmic transporter, as a factor necessary for the survival of Trp53-KO AML cells. sgRNAs targeting Xpo7 were enriched in Trp53-WT AML cells after the 16-day culture (Yamauchi et al. Cancer Cell 2018), while they were significantly depleted in Trp53-KO cells. Trp53-KO cells were vulnerable to Xpo7 depletion, while Xpo7 functioned as a Trp53-dependent tumor suppressor in Trp53-WT AML cells. As expected, either CRISPR/Cas9-mediated Xpo7 depletion or shRNA-mediated Xpo7 knockdown significantly delayed cell cycle progression and suppressed cell growth only in Trp53-KO AML cells, but not in WT cells. These findings were also validated using the DepMap (https://depmap.org), a publicly-available CRISPR/Cas9 dropout screen dataset. Significantly, XPO7 dependency was most correlated with that of TP53 and particularly evident in TP53-WT cell lines. We next performed CRISPR-Cas9 saturation mutagenesis scan targeting all Xpo7 exons using Trp53-WT and -KO mouse AML cells. Strikingly, sgRNAs targeting Xpo7 coding regions were significantly enriched only in the Trp53-WT cells, namely in the presence of an MDM2 inhibitor, supporting the notion that Xpo7 could function as a tumor suppressor when Trp53 function is intact and evident. In a stark contrast, the same sgRNAs, which target coding exons, were mostly depleted in Trp53-KO AMLs. Importantly, sgRNAs targeting the Xpo7 UTRs were unchanged/neutral regardless of experimental conditions (e.g. Trp53 status, MDM2 inhibitor treatment), serving as valid control for the experiments. Since Xpo7 presumably mediates the nuclear import and export of proteins, we hypothesized that Xpo7 promotes retention of Trp53 protein in the nucleus in Trp53-WT AML cells. As expected, Trp53 protein levels in the nucleus were significantly decreased, while those in the cytoplasm were increased upon Xpo7 depletion, revealed by Western blot. These data suggest that Xpo7 retains WT-Trp53 in the nucleus and functions as a Trp53-dependent tumor suppressor in Trp53-WT AML. Finally, to explore functional significance of XPO7 in human AML, we assessed correlation between XPO7 expression levels and AML subtypes and/or genetic background using publicly-available datasets. XPO7 mRNA levels were significantly upregulated in TP53-mutated AMLs in the TCGA datasets (Ley et al. NEJM 2013). XPO7 mRNAs levels were remarkably high in acute erythroid leukemia (AEL) cases, where TP53 mutations are frequently observed (Tyner et al. Nature 2018 and Iacobucci et al. Nat Genet. 2019). In fact, shRNA-mediated XPO7 knockdown significantly suppressed proliferation of HEL, a human AEL cell line harboring TP53 mutation. In summary, we identified a synthetic lethal relationship between TP53 and XPO7. Our study may facilitate the development of novel therapeutic strategies for TP53-mutated AML, such as AEL. Disclosures Akashi: Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding; Sumitomo Dainippon, Kyowa Kirin: Consultancy.