MYC Protein Expression Research Articles

HUWE1 Inhibition As a Therapeutic Strategy to Target MYC in MM

BackgroundThe use of proteasome inhibitors to disrupt the ubiquitin proteasome system (UPS) has provided a significant therapeutic advance and highlights the importance of this pathway in Multiple Myeloma (MM). There is increasing interest in developing therapeutic strategies to target enzymes that regulate the UPS, with the aim of gaining more specificity and reducing side-effects. We previously identified elevated expression of the E3 ligase HUWE1 in MM compared to normal cells and demonstrated that inhibition of HUWE1 with shRNA results in growth arrest of MM cell lines (Blood 2016, 128:240). HUWE1 is involved in the regulation of MYC and its expression is deregulated in a number of tumor types. The aim of this study was to elucidate the role and therapeutic potential of inhibiting HUWE1 in MM using small molecule inhibitors.MethodsInhibitors of HUWE1 (BI8622, BI8626) as described in Peter et al. (EMBO Mol Med 2014, 6:1525-1541) were purchased from Syngene. The effect of the inhibitors on MM cell lines (OPM-2, KMS-18, JJN3, MM.1S, MM.1R, ANBL6.WT, ANBL6.BR), patient cells and normal bone marrow (NBM) from healthy donors was assessed using CellTitre-glo and WST1 assays. Cells were analysed for cell cycle status (propidium iodide and EdU staining), apoptosis and β-galactosidase activity. Snapshot Proteomics (AVM Biomed) was performed on HUWE1 knockdown cells to identify novel ubiquitinated substrates of HUWE1. Signalling pathways and substrates of HUWE1 were analysed using UbiQapture-Q (Enzo Life Sciences), Western blotting and RQ-PCR. Calcusyn software was used to calculate a combination index (CI) to assess synergistic anti-MM activity.ResultsUbiQapture assays demonstrated that BI8622 and BI8626 blocked auto-ubiquitination of HUWE1 in a concentration-dependent manner, with an associated decrease in the ubiquitination of MIZ1 (a validated HUWE1 substrate). BI8622 and BI8626 significantly reduced the growth of MM cell lines, including those resistant to dexamethasone (MM.1R) and bortezomib (ANBL6.BR), and patient-derived MM cells (p ≤ 0.01; IC50 range 9-20 µM) without significantly affecting NBM, suggesting a favourable therapeutic index. Furthermore, both compounds inhibit the proliferation of MM cell lines when co-cultured with MM patient-derived bone marrow stromal cells. HUWE1 inhibition induced cell cycle arrest, associated with a reduction of cells undergoing DNA synthesis (p = 0.005), cellular senescence demonstrated by β-galatosidase staining and increased p16 protein expression. HUWE1 inhibition in MM cell lines resulted in a significant decrease in MYC mRNA and protein expression (p < 0.001), accompanied by downregulation of multiple MYC-dependent target genes (CCND2, HSPE1, ODC1, CCNA2, CDC25A; p ≤ 0.02). Proteomic profiling along with ingenuity pathway analysis revealed novel putative substrates of HUWE1 involved in the regulation of MYC. Finally, the combination of HUWE1 inhibitors with proteasome inhibitors (carfilzomib and bortezomib) and dexamethasone results in synergistic anti-MM activity (CI range 0.57 - 1).ConclusionsMYC is frequently dysregulated in MM and represents an attractive therapeutic target. Here we demonstrate that inhibitors of the E3 ligase HUWE1 lead to growth arrest of MM cell lines, associated with decreased expression of MYC and MYC-dependent target genes. While BI8622 and BI8626 have previously been reported to repress MYC activity in models of colorectal cancer, we demonstrate for the first time that in MM this is accompanied with a decrease in MYC expression. Studies are ongoing to elucidate the molecular mechanism of MYC downregulation following HUWE1 inhibition both in vitro and in vivo. In summary, this study demonstrates that HUWE1 is important in the growth and survival of MM cells and presents a novel therapeutic strategy for the inhibition of MYC function in MM. DisclosuresNo relevant conflicts of interest to declare.

DLBCL-Derived NF-κb Mutations Exploit Senescence and Immune Evasion Mechanisms to Promote Myc-Driven Lymphomagenesis

Introduction: Constitutive NF-κB activation is a hallmark feature of B-cell malignancies, including the ABC-subtype of diffuse large B-cell lymphoma (DLBCL), and is mainly due to gain-of-function mutations in components of the Toll-like receptor (MYD88) or B-cell receptor (CD79B, CARD11) pathways, inactivating mutations of cellular NF-κB suppressors (TNFAIP3/A20, BIRC3) or copy number gain (NFKBIZ), often in the presence of high Myc oncoprotein expression. In order to understand whether such genetic lesions may cooperate with Myc during lymphomagenesis, we took advantage of the Eµ- myc transgenic mouse in which aggressive B-cell lymphomas are induced by B-cell-specific overexpression of c-Myc.Methods: Eµ- myc transgenic hematopoietic stem cells (HSC) were stably transduced with GFP-expressing retroviral vectors encoding the murine orthologs of human MYD88-L265P, NFKBIZ, CD79B-ITAM-Y196H, CARD11-L244P, CARD11-L225LI or shRNA against TNFAIP3/A20 or BIRC3 prior to their transplantation into lethally irradiated syngenic C57BL/6 mice. Recipient animals were subsequently monitored for the development of a palpable lymphadenopathy, and lymphomas were subject to immunophenotyping, analysis of growth parameters and transcriptome profiling by RNA-Seq.Results : Ectopic overexpression of NFKBIZ, MYD88-L265P, CD79B-ITAM-Y196H or knockdown of TNFAIP3/A20 in Eµ- myc transgenic HSC gave rise to GFP(+) B-cell lymphomas (> 70% GFP+/B220+ cells), albeit at varying frequencies. All mutant-driven lymphomas manifested with reduced apoptotic TUNEL/B220 indices when compared to the empty-vector control cohort (ctrl. lymphomas). In contrast, neither knockdown of BIRC3 nor overexpression of the CARD11 mutants generated GFP(+) Eµ- myc lymphomas in vivo . Unexpectedly, we found senescent GFP(+) B- lymphocytes residing in the spleens of these healthy animals after transplantation. Genetic ablation of the senescence program (via allelic deletion of the histone demethylase Suv39h1 or the INK4a/ARF locus) in Eµ- myc HSC prior to transduction with the CARD11 mutants gave rise to palpable GFP(+) B-cell lymphomas (unlike the empty-vector), indicating that CARD11 missense mutations require a senescence-deficient background to promote Eµ- myc -driven lymphomagenesis.RNA-Seq unvealed downregulation of MHC class II genes as a common feature of all mutant-driven Eµ- myc lymphomas when compared to ctrl. lymphomas. Since MHC/T-cell receptor interactions are required for an effective anti-tumoral adaptive immune response, this finding suggested immune evasion mechanisms to be exploited by all genetic lesions tested to promote Myc-driven lymphomagenesis. Further supporting this notion, MYD88-L265P-driven lymphomas exhibited elevated surface expression of the immune checkpoint ligands PD-L1 and PD-L2. Strikingly, knockdown of PD-L1 alone was sufficient to significantly delay MYD88-L265P-driven lymphomagenesis in Eµ- myc HSC transplantation experiments, suggesting that PD-L1-mediated immune evasion mechanisms contribute significantly to the aggressive phenotype observed under the MyD88-L265P mutant in vivo . Furthermore, overexpression of PD-L1 in Eµ- myc HSC alone also induced GFP(+) B-cell lymphomas that displayed a striking overlap in gene expression with MyD88-L265P-driven Eµ- myc lymphomas.Conclusions: We continue to characterize the Eµ- myc transgenic mouse as a well-suited model platform to functionally dissect DLBCL-relevant oncogenic networks in general and NF-κB-activating mutations in the context of high Myc protein expression, a frequent feature of DLBCL, in particular. Our systematic in vivo analysis of different NF-κB-activating mutants - so far rather recognized as functionally interchangeable moieties - indicated that some of these genetic lesions directly cooperate with Myc (i.e. MyD88-L265P, NFKBIZ) - partly via utilizing immune evasion mechanisms - while others (e.g. CARD11 mutations) require upfront senescence-ablating lesions to license Myc-driven lymphomagenesis. Our findings might explain the pressure to select for senescence-ablating lesions like INK4a/ARF loss frequently found in human DLBCL, and could inform immune checkpoint blockade treatment strategies based on certain oncogenic/immune evasion network interconnections. DisclosuresNo relevant conflicts of interest to declare.

Stratification By MYC Protein Expression Level Improves the Prognostic Impact of MYC Translocation in Patients with Large B-Cell Lymphoma

Introduction: In patients with Large B-cell Lymphoma (LBCL) the prognostic significance of MYC translocations is not clearly established. We aimed to investigate if additional identification of MYC translocation partner gene and/or MYC and BCL2 protein expression levels could improve patient prognostication associated with MYC translocations. We also wanted to evaluate the prognostic effect of double hit MYC + BCL2/BCL6 translocation (DH) compared to MYC single hit (SH).Methods: A consecutively collected training cohort of 64 patients and an independent validation cohort of 28 patients all with MYC translocation positive LBCL were investigated. Translocations were identified with fluorescent in situ hybridization (FISH) and MYC and BCL2 protein expression with immunohistochemistry (IHC).Results: MYC> 75% was seen in 50% of patients and was related to frequent bone marrow involvement, high IPI and elevated LDH. MYC>75% was also significantly associated with reduced progression free survival (PFS) and overall survival (OS) in univariate analysis (PFS: HR 6.8 (95%CI 1.5-31), p=0.004. OS: HR 4.3 (95%CI 0.9-21), p=0.05) (Fig. 1). In multivariable analysis adjusting for IPI MYC>75% was independently prognostic for PFS but not OS (PFS: HR 5.4 (95%CI 1.1-25), p=0.04. OS: HR 3.1 (95%CI 0.6-16), p=0.2). DH was seen in 71% of patients and did not confer a worse outcome compared to MYC SH. Immunoglobulin (IG) MYC translocation partner gene was found in 55% and was related to MYC protein expression levels (p=0.047). Translocation partner gene was not prognostic for PFS (p=0.8) or OS (p=0.6). High BCL2 expression was related to high MYC expression (p=0.003) and did not add to the prognostic effect observed for MYC. The findings were confirmed in a comparable, independent validation cohort of 28 MYC translocation positive LBCL. The patients included in the survival analyses were treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) or R-CHOEP (R-CHOP + etoposide).Conclusions: These findings suggest that stratification by MYC protein expression level significantly improves the prognostic impact of MYC translocation in patients with large B-cell lymphoma. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

RNF8 stabilizes MYC via K63‐linked ubiquitination to promote metabolic reprogramming in triple‐negative breast cancer

Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer owing to its aggressive nature and the lack of targeted therapy for most patients. MYC, an oncogenic transcription factor, is elevated in TNBC and causes accelerated tumor growth through metabolic reprogramming. However, there is currently no drug available in clinics to inhibit MYC. Ring Finger Protein 8 (RNF8), an E3 ubiquitin ligase, is highly expressed in TNBC and essential for tumor progression. To comprehensively understand the role of RNF8 in TNBC, we conducted RNA-sequencing and found that the downregulation of RNF8 attenuates the activity of the MYC network in human TNBC cell lines. Moreover, we observed the reduced glucose uptake and utilization in TNBC cells with RNF8 knockdown as a result of the decreased expression of MYC and its target genes such as glucose transporter GLUT1 and key glycolytic enzyme HK2. Knockdown of RNF8 also decreased the metabolic influx in the glycolysis pathway as revealed in the isotope tracer studies coupled with mass spectrometry analysis. Importantly, the treatment of proteasome inhibitor MG132 was able to restore MYC protein expression, while cycloheximide chase assay showed that MYC protein has a shorter half-life in TNBC cells with RNF8 deficiency. In vivo ubiquitination assay further demonstrated that RNF8 mediates Lysine (K)63-linked ubiquitination over MYC, suggesting that RNF8 mediates K63-linked ubiquitination to stabilize MYC against proteasomal degradation. Taken together, these results suggest that RNF8 enhances MYC signaling, thereby promoting metabolic reprogramming and tumor progression in TNBC. Targeting RNF8 may be a reasonable alternative to inhibit MYC-driven cancers such as TNBC.

MYC protein expression does not correlate with MYC abnormalities detected by FISH but predicts an unfavorable prognosis in de novo acute myeloid leukemia

While dysregulation of MYC has been implicated in acute myeloid leukemia (AML), the impact of MYC protein expression in AML is less well understood. We investigated the correlation of MYC protein expression by immunohistochemistry (MYC-IHC) with MYC abnormalities and prognosis in adult de novo AML. MYC-IHC in bone marrow of patients with untreated AML (n = 58) was assessed and scored as MYClow (0–40 % of blasts) or MYChigh (> 40 % of blasts). This was correlated with MYC abnormalities by fluorescence in situ hybridization (FISH) and prognosis in the context of cytogenetic risk stratification. Residual myeloid disease at the end of induction was assessed by flow cytometry. MYClow and MYChigh were detected in 24 (41 %) and 34 cases (59 %), respectively. Extra copies of MYC were present in 12 % of cases and were not correlated with level of MYC-IHC. No cases had MYC translocation or amplification. Compared to MYClow patients, MYChigh patients had a shorter overall survival in all cytogenetic risk groups (68 vs. 21 months, p = 0.006) and in the intermediate risk group (61 vs. 21 months, p = 0.046). MYChigh patients had a tendency towards detected residual disease at the end of induction in all cytogenetic risk and intermediate risk groups. Regardless of the underlying mechanisms of MYC dysregulation, high level of MYC protein is expressed in the majority of AML and correlated to worse prognosis. Further studies on MYC dysregulation in leukemogenesis and therapy targeting MYC aberration are warranted.

Lack of expression of LMO2 clone SP51 identifies MYC rearrangements in aggressive large B-cell lymphomas.

MYC rearrangements (MYC-R) confer unfavorable prognosis to large B-cell lymphomas (LBCL). Because of the low incidence of such genetic alteration, surrogates to screen MYC-R may be useful in daily practice. Previous studies suggested that clone 1A9-1 of LMO2 loss may be a good predictor for the presence of MYC-R in LBCL. The present study examines the utility of LMO2 clone SP51. For this purpose, we have analyzed 20 Burkitt lymphomas and 325 LBCL. Among them, 245 cases were studied prospectively using whole tissue sections, and 100 retrospectively by tissue microarrays. The cohort of CD10-positive prospective cases achieved the best results. Lack of LMO2 SP51 expression predicted the presence of MYC-R with high specificity, accuracy, positive and negative predictive value (PPV/NPV), and positive and negative likelihood ratios (PLR/NLR). Compared with MYC protein expression, LMO2 SP51 obtained significantly higher specificity, accuracy, PPV, and PLR (94%, 91%, 85%, and 14.33 vs 73%, 77%, 56%, and 3.26, respectively), and similar NPV and NLR (92% and 0.22 vs 95% and 0.12). Compared with LMO2 clone 1A9-1, the sensitivity of LMO2 SP51 was lower (79% vs 89%). We conclude that LMO2 SP51 may be a useful marker to screen MYC-R in CD10-positive LBCL.

The combination of C-Myc rearrangement and 1q21 gain is associated with poor prognosis in multiple myeloma

The prognostic value of chromosomal 1q21 gain in newly diagnosed multiple myeloma (NDMM) remains controversial. Add-on Myc aberrations may further worsen the outcome. To investigate whether specific genes located at the 1q21 region, such as myeloid cell leukemia 1 (Mcl-1), are involved in NDMM progression, we examined bone marrow cytogenetic abnormalities in 153 patients with NDMM by fluorescence in situ hybridization. Their response to treatment and survival was also analyzed. C-Myc and Mcl-1 expressions in bone marrow samples were analyzed by RT-PCR. The expression of Mcl-1 was evaluated in bone marrow sections by immunohistochemistry. MM cell lines were transfected with Mcl-1 siRNA. 1q21 gain was present in 55/153 (35.9%) patients and strongly associated with Myc rearrangement (31/153, 20.3%, P = 0.004). A positive correlation was observed between Myc and Mcl-1 mRNA levels in bone marrow cells from 47 patients (r = 0.57, P < 0.001). The combination of 1q21 gain and Myc rearrangement was associated with poorer overall survival than Myc rearrangement alone (16.8 vs. 27.9 months, P = 0.077) or 1q21 gain alone (16.8 vs. 60.7 months, P < 0.01). High Mcl-1 protein expression in bone marrow plasma cells was associated with Myc rearrangement. Mcl-1 silencing by siRNA inhibited Myc protein expression in three myeloma cell lines. Treatment with the small-molecule Mcl-1 inhibitor, UMI-77, produced similar results. Overall, the combination of Myc rearrangement and 1q21 gain was associated with particularly poor prognosis in patients with MM. Furthermore, our data are consistent with Mcl-1-dependent Myc protein activation.

Crizotinib Resistance Mediated by Autophagy Is Higher in the Stem-Like Cell Subset in ALK-Positive Anaplastic Large Cell Lymphoma, and This Effect Is MYC-Dependent

Simple SummaryAutophagy is a cell survival and recycling mechanism which protects cancer cells upon therapeutic drug treatment. Here we investigated the impact of autophagy inhibition in a cancer of lymphoid origin, namely ALK-positive anaplastic large cell lymphoma (ALK + ALCL). We inhibited autophagy in two distinct cell subsets of ALK + ALCL, one of which we previously shown to possess more stem-like and tumorigenic properties. Our study found that blockage of autophagy in the stem-like subset resulted in marked drug-sensitization to crizotinib, a current therapeutic agent used to treat ALK + ALCL. We also found differential involvement of the Myconcogene in the autophagy process within the two subsets and identified its relative importance to the stem-like population. Our research suggests inhibition of autophagy alongside crizotinib preferentially targets stem-like cells, thus improving crizotinib therapy.Previously it was shown that autophagy contributes to crizotinib resistance in ALK-positive anaplastic large cell lymphoma (ALK + ALCL). We asked if autophagy is equally important in two distinct subsets of ALK + ALCL, namely Reporter Unresponsive (RU) and Reporter Responsive (RR), of which RR cells display stem-like properties. Autophagic flux was assessed with a fluorescence tagged LC3 reporter and immunoblots to detect endogenous LC3 alongside chloroquine, an autophagy inhibitor. The stem-like RR cells displayed significantly higher autophagic response upon crizotinib treatment. Their exaggerated autophagic response is cytoprotective against crizotinib, as inhibition of autophagy using chloroquine or shRNA against BECN1 or ATG7 led to a decrease in their viability. In contrast, autophagy inhibition in RU resulted in minimal changes. Since the differential protein expression of MYC is a regulator of the RU/RR dichotomy and is higher in RR cells, we asked if MYC regulates the autophagy-mediated cytoprotective effect. Inhibition of MYC in RR cells using shRNA significantly blunted crizotinib-induced autophagic response and effectively suppressed this cytoprotective effect. In conclusion, stem-like RR cells respond with rapid and intense autophagic flux which manifests with crizotinib resistance. For the first time, we have highlighted the direct role of MYC in regulating autophagy and its associated chemoresistance phenotype in ALK + ALCL stem-like cells.

Evaluation of MYC protein expression using immunohistochemistry in extranodal Diffuse large B-cell lymphoma

Evaluation of MYC protein expression using immunohistochemistry in extranodal Diffuse large B-cell lymphoma

RNA-Based next generation sequencing complements but does not replace fluorescence in situ hybridization studies for the classification of aggressive B-Cell lymphomas

Aggressive B-cell lymphomas are currently classified based in part upon the presence or absence of translocations involving BCL2, BCL6, and MYC. Most clinical laboratories employ fluorescence in situ hybridization (FISH) analysis for the detection of these rearrangements. The potential role of RNA-based sequencing approaches in the evaluation of malignant lymphoma is currently unclear. In this study, we performed RNA sequencing (RNAseq) in 37 cases of aggressive B-cell lymphomas using a commercially available next generation sequencing assay and compared results to previously performed FISH studies. RNAseq detected 1/7 MYC (14%), 3/8 BCL2 (38%) and 4/8 BCL6 (50%) translocations identified by FISH. RNAseq also detected 1 MYC/IGH fusion in a case not initially tested by FISH due to low MYC protein expression and 2 BCL6 translocations that were not detected by FISH. RNAseq identified the partner gene in each detected rearrangement, including a novel EIF4G1-BCL6 rearrangement. In summary, RNAseq complements FISH for the detection of rearrangements of BCL2, BCL6 and MYC in the evaluation and classification of aggressive B-cell lymphomas by detecting rearrangements that may be cryptic by FISH methods and by identifying the rearrangement partner genes. Detection of these clinically important translocations may be optimized by combined use of FISH and RNAseq.

MYC gene amplification by fluorescence in situ hybridization and MYC protein expression by immunohistochemistry in the diagnosis of cutaneous angiosarcoma: Systematic review and appropriate use criteria.

Secondary angiosarcoma (AS) most commonly follows breast cancer and includes postirradiation AS (PRAS) and lymphedema-associated AS. The frequent amplification of MYC (8q24.21) in secondary AS and the rising incidence of PRAS and atypical vascular lesions (AVLs) have prompted interest in the diagnostic and prognostic utility of MYC in AS. Retrospective series with ≥2 cases of cutaneous AS and describing the use of fluorescence in situ hybridization (FISH) for MYC amplification or immunohistochemistry (IHC) for MYC overexpression were included. Sixteen studies met inclusion criteria. Overall, 93% of cases evaluated by FISH and IHC were concordant. The sensitivity of FISH in primary AS was only 6.8%, and protein overexpression occurred without amplification in sun-damaged skin. FISH and IHC were over 78% sensitive in secondary AS but negative in over 98% of AVLs. MYC amplification and FLT4 coamplification were associated with shorter overall survival in secondary AS. FISH for MYC amplification and IHC for MYC overexpression are useful in distinguishing PRAS from AVLs and may also have prognostic value in secondary AS. In contrast, these methods have little diagnostic or prognostic value in primary AS and should not be used to distinguish primary AS from benign vascular neoplasms.

IgM-Secreting Diffuse Large B-Cell Lymphoma (DLBCL) Is a Poor Prognostic Subset within the Non-Germinal-Centre-Type (GC-type): An Italian Multicentre Study

BACKGROUND: In 2014 we identified a new subset of DLBCL, defined as "IgM-secreting" (Cox MC &amp; Di Napoli A , PLOS One 2014). This was characterised by poor prognostic features and outcome as well as frequent central nervous (CNS) system localizations. Furthermore, IgM-secretion, was an independent prognostic factor in multivariate analysis. Here we report on the largest series of IgM-secreting-DLBCL, from a multicentre Italian study. METHODS: The observational and biological study was approved by the Ethical Committee of the AUO Sant'Andrea, Italy. Enrolment criteria were: DLBCL with an associated IgM paraprotein diagnosed between 1st January 2010 and 31st December 2018 (IgM-secreting). Data were collected both prospectively and retrospectively from 17 Centres participating in the study. In addition, histopathology samples were centrally revised for immunohistochemistry (IHC) and FISH analyses. The control group (CTRL) consisted in a series of consecutive DLBCL, without an associated IgM-paraprotein (diagnosed between 01/01/2013 and 30/06/2016, enrolled in the Lymphoma Registry of the Lazio region (ReLLi Network). Last follow-up was carried out on 31st December 2019. RESULTS: 569 DLBCL cases were enrolled: 102 (17.9%) were IgM-secreting; 48 (8.4%) had a non-IgM paraprotein (IgA, IgG, or other), and 414 (72.7%) had no associated paraprotein (CTRL). IgM-secreting cases within the consecutive DLBCL patients enrolled in the ReLLi Registry were 41/466 (8.8%, 95CI 6.4-11.7%) while non IgM-paraprotein DLBCL cases were 11/466 (2.4%, 95CI 1.2-4.2%). The median level of IgM paraprotein was 17gr/L (range: &amp;lt;1-84gr/L); 83/102 (81.3%) were IgMk and 23/102 (22.5%) IgML respectively. The IgM-secreting group differed from the CTRL because the following characteristics were significantly more frequent: 1] age&amp;gt;60 (p=.001); 2] advanced stage (p&amp;lt;.001); 3] PS≥2 (p=.001); 4] LDH&amp;gt;UNL (p=.008) ; 5] ≥2 Extra-nodal sites involved (p&amp;lt;.001) ; 6] IPI 3-5 (p&amp;lt;.001); 7] central nervous system (CNS) involvement at diagnosis or relapse (p&amp;lt;.001); 8] lower rate of complete remission(CR) at the end of induction immunochemotherapy (p&amp;lt;.001). Conversely, no differences were observed for: sex, B-symptoms, HCV and HBV status, bulky disease, age≥80 years, and for transformation from low-grade lymphoma. PATHOLOGICAL AND MOLECULAR FEATURES: Paraffin tissue from 74 CTRL and 69 IgM-secreting was suitable for immunohistochemistry (IHC). The non-GCB subtype, based on Hans algorithm, was prevalent in the IgM-secreting (p=.005). No difference in BCL2 expression alone or in MYC and BCL2 double expression was observed within groups. In 48/63(76%; 95CI: 64-86%) IgM-secreting cases, both the IgM heavy and the corresponding kappa or lambda light chain protein expression were detected in the cytoplasm of the neoplastic clone. FISH analyses for MYC, BCL2 and BCL6 genes rearrangements performed in 25 IgM-secreting cases with either expression of MYC protein or a GC-phenotype showed no evidence of double or triple hits (DH/TH). TREATMENT: in the IgM-secreting group more patients were treated with RCOMP and with less intensive approach than the CTRL (p&amp;lt;.001). SURVIVAL: The median follow-up time was 46 months (95CI= 44-49; range 18-101) with 130 events and an incidence rate x100 person/year of 7.22 (95%CI 6.08-8.58) and a 5-yr OS of 76% (95CI 72-79%). The 5-PFS was 61% (95CI 57-65%). In univariable analysis age&amp;gt;60, B-symptoms, bulky disease, IPI &amp;gt;low risk and IgM-secreting IgM showed a worse survival (all with p&amp;lt;0.001). Also, the IgM-secreting group, showed a worse survival compared to the DLBCL with an associated IgG/IgA paraprotein (p&amp;lt;0.001). Adjusting in multiple Cox regression, IgM-secreting with IPI, gender, bulky and B-symptoms, maintain a higher risk of death either in the all cohort (HR 1.93, 95CI 1.34-2.78, p&amp;lt;0.001) or in patients with age&amp;lt;80 (HR 1.71, 95CI 1.16-2.54, p=0.007). Noteworthy, a survival sub-analysis showed that the 12/69 (17.4%) IgM-secreting with a GC-type had a better OS (9=0.008) and PFS (p=0.002) compared to the 57/69 (82.6%) IgM-secreting with a non-GC-type. CONCLUSION: Our data confirm that IgM-secreting DLBCL: 1) represents a sizable proportion of non-DH DLBCL; 2) have poor prognostic features and 3) have mostly a non-GC phenotype. Furthermore, IgM secretion appears to be an independent prognostic factor for both PFS and OS. Studies to define the biological features of this new subset are ongoing. Disclosures Cantonetti: Mundipharma: Consultancy; Takeda: Consultancy; Vifor: Consultancy; Roche: Consultancy. Re:BerGenBio ASA: Research Funding. Abruzzese:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bms: Honoraria.

High MYC Protein Expression in MDS Is Associated with Early Transformation to AML

Acute myeloid leukemia (AML) with myelodysplasia-related changes (AML-MRC) is shown to be associated with dismal clinical outcomes and there remains a pressing need for new therapeutic strategies to improve survival outcomes. It is also important to identify targetable markers that could predict an earlier AML transformation from preexisting MDS. Our recent studies demonstrated that high expression level of MYC oncoprotein is associated inferior survival outcome in AML-MRC patients and that MYC plays an oncogenic role by epigenetic regulation of hydroxylation of 5-methylcytosine. However, the role of MYC in MDS to AML progression remain to be answered. Here, we investigated MYC protein expression levels in bone marrow (BM) specimens of patients with preexisting MDS and their subsequent AML-MRC specimens to determine whether increased MYC expression is associated with early AML progression. We retrospectively identified 32 patients with histologically confirmed AML-MRC evolving from MDS and evaluated MYC protein levels in BM biopsy specimens at the time of initial MDS diagnosis and at the time of disease transformation to AML-MRC, respectively. Clinical data including age at diagnosis, gender, CBC with differential count, cytogenetics, somatic mutations common in myeloid disease, blasts count, and IPSS were extracted at the time of MDS and AML-MRC diagnosis. MYC expression was assessed by immunohistochemistry on 4-5µm tissue sections. Deparaffinized slides were stained with anti-MYC antibody (clone Y69, Roche Diagnostics) using a Ventana Benchmark automated system. MYC expression was scored independently by two hematopathologists and categorized as low vs. high as previously described. The cut-off for high MYC expression is ≥5% positive cells. The MDS-to-AML progression free survival (PFS) and overall survival (OS) were calculated from the date of MDS diagnosis and outcomes were estimated with the Kaplan-Meier method and compared using the log-rank test. All statistical analyses were performed using SPSS v24.0. Among 32 patients, 69% (n=22) and 31% (n=10) patients had low and high MYC expression at MDS diagnosis (Table 1). The median age was 67.9 (33.5-78.7) and 61.1 (38.9-73.4) and the % of MYC positive blasts were 1% (range, 0-4.5) and 17.5% (range, 5-27.5) in low and high MYC groups, respectively, at the time of MDS diagnosis. There was no statistical difference in BM blast counts between low vs. high MYC groups (median 5% vs. 7.5%, p=0.4951). IPSS risk assessment in a total of 26 patients (19 in low MYC and 7 in high MYC) showed that 9% (n=2), 27% (n=6), 32% (n=7), and 18% (n=4) in low MYC group and 0% (n=0), 20% (n=2), 20% (n=2), and 30% (n=3) in high MYC groups had low, intermediate-I, intermediate-II, and high risk at the time of MDS diagnosis (Table 1). A total of 84% (86% [n=19] in low MYC and 80% [n=8] in high MYC) patients received treatments for MDS; ESA in 16% (n=5), hypomethylating agents in 81% (n=26), and Revlimid in 16% (n=5) (Table 1). Allogeneic stem cell transplant (allo-SCT) was performed in 12.5% (n=4) patients (n=3, low MYC; n=1, high MYC). Following AML progression, a total of 91% (n=29) patients received treatment for AML including induction chemotherapy (n=18), hypomethylating agents (n=7), and other therapies including trial (n=4) (Table 1). In the univariate analyses, patients with low MYC expression had significantly longer MDS-to-AML PFS (median 15.8 vs. 7.25 months, HR=0.1745, 95%CI=0.0591-0.5157, p=0.0016) (Figure 1), however, there was no OS difference between two groups (4.32 vs. 2.35 years, HR=0.5131, 95%CI=0.1064-2.475, p=0.6909). In a multivariate Cox model (adjusting for IPSS, age, front-line therapy, gender, allo-SCT, and MYC level), high MYC expression (HR=3.939, 95%CI=1.107-14.012, p=0.034) and IPSS risk (HR=1.903, 95%CI=1.139-3.181, p=0.014) were significant factor for MDS-to-AML PFS. In conclusion, high MYC expression at the MDS stage was associated with shorter time to AML progression, indicating that MYC plays an oncogenic role in MDS to AML transformation. Although our findings need further validation by prospective studies with greater sample size, our results support a combination treatment of hypomethylating agents with novel agents targeting MYC or/and its downstream pathways and warrant further study to identify underlying mechanisms of MYC-driven MDS to AML progression. Disclosures Talati: Pfizer: Honoraria; Astellas: Speakers Bureau; Jazz: Speakers Bureau; BMS: Honoraria; AbbVie: Honoraria. Kuykendall:Blueprint Medicines: Research Funding; BMS: Research Funding; Incyte: Research Funding; Novartis: Research Funding. Padron:Novartis: Honoraria; Incyte: Research Funding; Kura: Research Funding; BMS: Research Funding. Komrokji:JAZZ: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Novartis: Honoraria; Incyte: Honoraria; Acceleron: Honoraria; Geron: Honoraria; AbbVie: Honoraria; Agios: Honoraria, Speakers Bureau. Lancet:Abbvie: Consultancy; Agios Pharmaceuticals: Consultancy, Honoraria; Astellas Pharma: Consultancy; Celgene: Consultancy, Research Funding; Daiichi Sankyo: Consultancy; ElevateBio Management: Consultancy; Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy. Sallman:Celgene, Jazz Pharma: Research Funding; Agios, Bristol Myers Squibb, Celyad Oncology, Incyte, Intellia Therapeutics, Kite Pharma, Novartis, Syndax: Consultancy.

Inhibition of PIM Kinases in Diffuse Large B-Cell Lymphoma Cells Targets MYC-Dependent Transcriptional Program, Increases CD20 Expression and Augments the Efficacy of Anti-CD20 Antibodies

R-CHOP immunochemotherapy remains standard frontline therapy for newly diagnosed diffuse large B-cell lymphoma (DLBCL) patients. However, this therapy is ineffective in approximately 1/3 of patients, underscoring the need for better treatment modalities. Targeting DLBCL oncogenic drivers is a promising strategy to improve the treatment efficacy and outcome. Although MYC transcription factor is one of the key oncogenes in DLBCL development, direct MYC targeting strategies have been largely ineffective, highlighting the need for other, indirect approaches. For example, MYC expression is stabilized by PIM serine-threonine kinases, indicating that PIM inhibition might be a rational approach to indirectly target MYC. In this study, we assessed the PIM-MYC relationship and the consequences of PIM inhibition in DLBCL. We first evaluated the expression of PIM1-3 and MYC proteins in 57 DLBCL diagnostic sections by immunohistochemistry. In this series, 70.17% of specimens were positive for at least one PIM isoform and 84.22% cases were MYC-positive. 100% of cases with high MYC expression (MYC present in ≥30% of the cells, n=35) were PIM-positive, whereas 86,36% of cases with undetectable or low MYC expression (MYC detected in ≤20% of cells, n= 22) were PIM-negative (Fisher's exact test, p&amp;lt;0.0001). Since the coexpression of MYC and PIMs highlights the functional link between these proteins in DLBCLs, we evaluated the expression of PIM kinases in cell lines following siRNA-mediated MYC knockdown or treatment with MYC-MAX dimerization inhibitor, 10058F4. The genetic or chemical MYC inhibition markedly decreased PIM1-3 expression in six GCB and ABC cell lines. Likewise, knockdown of all three PIM isoforms decreased MYC levels, attenuated proliferation and induced apoptosis. Similarly, PIM blockade with SEL24/MEN1703, a novel pan-PIM/FLT3 inhibitor tested currently in clinical trial in AML patients and exhibiting favorable safety profile, decreased the expression of multiple MYC-dependent genes. To assess the MYC role in PIM inhibitor-mediated toxicity, we generated DHL4 cells expressing degradation-resistant MYC_T58A mutant. MYC_T58A expression partially protected cells from PIM inhibitor-induced proliferation arrest and apoptosis, indicating that the inhibitor's toxicity is at least partially mediated by MYC depletion. The MS4A1 gene, encoding CD20 surface antigen and rituximab target, is regulated by an upstream promoter containing potential MYC-binding sites (E-boxes). MYC association to these regions was confirmed in chromatin immunoprecipitation assays. As expected, in SEL24/MEN1703-treated cells, MYC occupancy at the MS4A1 promoter markedly decreased. To determine the consequences of MYC binding to the MS4A1 promoter, we assessed CD20 levels in a lymphoblastoid cell line carrying tetracycline-regulated (tet-off) MYC. MYC repression markedly elevated transcript and surface CD20 levels in a time-dependent manner, reaching 17.3-fold (transcript) and 3.82-fold (surface) inductions at 96 h. Consistently, the pan-PIM inhibitor decreased MYC expression in DHL4 and RAJI cells and resulted in increased surface CD20 levels up to 3.72-fold of baseline. In cells expressing the MYC_T58A mutant, PIM inhibition did not increase CD20 level, indicating that PIM kinases modulate CD20 surface expression via MYC. Importantly, PIM inhibitors increased CD20 levels also in primary, patient-derived DLBCL cells. These data suggest that indirect MYC targeting via PIM inhibition would lead to increased rituximab activity. Indeed, in PIM inhibitor-treated DHL4 and RAJI cells, rituximab triggered higher complement-dependent toxicity. Likewise, PIM inhibitor potentiated rituximab-dependent uptake of DHL4 and DHL6 cells by human monocyte-derived macrophages in antibody-dependent cellular phagocytosis assay. Taken together, we characterize a PIM-MYC regulatory circuit promoting DLBCL growth and resistance to anti-CD20 antibody. We also demonstrate that PIM inhibition exhibits pleiotropic effects that combine direct cytotoxicity with increased surface CD20 levels and increased susceptibility to anti-CD20 antibody-based therapies. Study supported by Foundation for Polish Science (POIR.04.04.00-00-5C84/17-00), Polish National Science Centre (2016/22/M/NZ5/00668 and 2017/26/D/NZ5/00561) and Ministry of Science and Higher Education in Poland (iONCO) grants. Disclosures Golas: Ryvu Therapeutics: Current Employment. Green:KDAc Therapeutics: Current equity holder in private company. Tomirotti:Menarini Ricerche: Current Employment. Brzózka:Ryvu Therapeutics: Current Employment. Juszczynski:Ryvu Therapeutics: Other: member of advisory board.

Autologous Vs. Allogeneic Stem Cell Transplantation in Double-Expressor Lymphoma

Purpose: Dual expression of MYC and BCL2 proteins (Double Expressor Lymphoma-[DEL]) is an important prognostic factor in patients (pts) with diffuse large B-cell lymphoma (DLBCL) who are treated with standard chemo-immunotherapy. Studies have suggested that pts with these biomarkers have inferior survival compared to pts who were non-DEL after autologous stem cell transplantation (autoSCT). For this reason, allogeneic stem cell transplantation (alloSCT) has been advocated for its potential graft-versus-lymphoma effect. Data are limited however regarding the outcomes in pts with relapsed DEL who were treated with autoSCT vs. alloSCT. Methods: Data from pts with relapsed DEL/DLBCL who underwent auto- and alloSCT as their first transplant at our center and in whom archived tumor material was available were analyzed. Cutoff values of 40% for MYC and 70% for BCL2 were established by immunohistochemistry (IHC). The majority of autoSCT pts (84%) underwent chemo-mobilization of stem cells with rituximab (R) for in-vivo purging; rituximab was also given on days+1 and +8 with BEAM conditioning (J Clin Oncol 2005; 23:2240-7; Clin Cancer Res 2018; 24:2304-11). Conditioning for the alloSCT was myeloablative (n=16, 50%), reduced-intensity (n=6, 19%), and nonmyelablative (n=10,31%). The study was IRB-approved at our center. Kaplan-Meier analysis and corresponding log-rank/Gray's tests were used to estimate and compare overall survival (OS), progression-free survival (PFS), and cumulative incidence (CI) of non-relapse mortality and relapse by SCT group. Standard multivariable Cox regression was used to evaluate associations between SCT type and OS/PFS after controlling for differences in baseline characteristics and cause-specific multivariable Cox regression was used for competing-risk outcomes [non-relapse mortality (NRM) and relapse]. Results: The study included 161 autoSCT and 32 alloSCT pts treated between 2000-2018. Median age was 59 (range,18-80) and 55 years (range, 21-66), respectively (P=0.009). Male gender was 62.7 vs 50.0%, respectively (P= 0.234). Fifty-one (32.5%) autoSCT pts and 9 (28.1%) alloSCT pts had an elevated serum LDH (P= 0.683) and 41/148 (27.7%) autoSCT and 11/25 (44.0%) alloSCT pts were PET-positive at study entry (P=0.106). The time from diagnosis to transplant was 20.0 and 25.2 months, respectively, (P=0.068), and the distribution of the years of transplant was similar between both groups (P=0.317). Significant differences between treatment groups were observed for prior number of chemotherapies, disease status, stage, and HCT-CI score at transplant. Pts. receiving alloSCT were more heavily pretreated [median 3 prior therapies, (range 1-8) vs. a median of 2, (range 1-6); P=0.0001], had more advanced stage III-IV disease (P=0.035), and more refractory disease (P=0.010) at transplant. In addition, a higher percentage of alloSCT pts had an HCT-CI of &amp;gt;4 (28.1% vs. 10.2%, P=0.018) at study entry. Allogeneic transplant characteristics included matched siblings (n=18; 56.3%), matched unrelated (n=13, 40.6%) and haplo-identical (n=1, 3.1%) donors. With a median follow-up for autoSCT surviving pts of 65 months (range, 5-217 months) and 93 months (range, 34--124 months) for alloSCT pts, the OS at 5-years were 59% and 34%, respectively (P= 0.0001) (Figure1). The PFS rates at 5-years were 49% and 31%, respectively (P = 0.002). AutoSCT had a similar rate of relapse to alloSCT but a lower rate of NRM. The 5-year CI of relapse was 37% vs 28%, respectively (P = 0.611) and the 5-year NRM rates were 14% vs 41 % (P=0.0001), respectively (Figure 2). The associations between SCT group and outcomes persisted after adjusting for differences between groups (alloSCT vs. autoSCT HR= 3.46/p=0.0004 for OS; HR=2.67/p=0.005 for PFS; HR=4.74/p=0.009 for NRM; HR=2.22/p=0.080 for relapse). The 100-day CI of grade II-IV and III-IV acute GVHD in the alloSCT group was 37.5 and 9.4%, respectively. The 1-year CI of chronic GVHD was 25.0%. C onclusions: This study is the first to show that autoSCT confers a superior survival in pts with relapsed DEL/DLBCL compared to alloSCT. Our conclusions are supported by long-term follow-up. The use of novel nonmyeloablative regimens in a larger number of pts receiving an alloSCT and ongoing studies at our center with targeted investigational agents after autoSCT may improve results. Disclosures Khouri: Pfizer: Research Funding; Bristol Myers Squibb: Research Funding. Iyer:CRISPR: Research Funding; Spectrum: Research Funding; Daiichi Sankyo: Consultancy; Trillium: Research Funding; Curio Biosciences: Honoraria; Target Oncology: Honoraria; Afffimed: Research Funding; Merck: Research Funding; Legend Biotech: Consultancy; Rhizen: Research Funding; Seattle Genetics, Inc.: Research Funding. Popat:Bayer: Research Funding; Novartis: Research Funding. Qazilbash:Angiocrine: Research Funding; Amgen: Research Funding; Bioclinica: Consultancy; Janssen: Research Funding; Bioline: Research Funding. Champlin:DKMS America: Membership on an entity's Board of Directors or advisory committees; Actinium: Consultancy; Johnson and Johnson: Consultancy; Omeros: Consultancy; Cytonus: Consultancy; Takeda: Patents &amp; Royalties; Genzyme: Speakers Bureau.

Socioeconomic Disparities in Diagnostic Testing Among Patients with Diffuse Large B-Cell Lymphoma in the US

Background A better understanding of diffuse large B-cell lymphoma (DLBCL) subtypes characterized by cell of origin (COO), molecular and immunophenotypic features is associated with more accurate prognosis and may guide treatment selection to achieve better outcomes. Current World Health Organization (WHO) and National Comprehensive Cancer Network (NCCN) guidelines recommend for all patients with DLBCL, immunohistochemistry (IHC, such as Hans algorithm) as a surrogate of gene expression profiling to identify COO, and molecular testing by fluorescence in situ hybridization (FISH) or karyotype to identify molecular/cytogenetic features. Despite advances in treatment and increasing knowledge of clinical importance of DLBCL subtype classification, socioeconomic disparities persist in the treatment of DLBCL in the US (Shenoy PJ, et al. Cancer 2011;117(11):2530-2540) and the use of guideline-recommended diagnostic testing in clinical practice remains unclear. Understanding whether test utilization varies by socioeconomic determinants is important to enable all patients to achieve better/equal access to health care and optimal outcomes. Methods To understand guideline-recommended diagnostic testing utilization in real-world settings and to assess socioeconomic disparities in relation to test utilization among patients with DLBCL in the US, this retrospective cohort study leveraged data from the Flatiron Health electronic health record (EHR)-derived de-identified database. Patients above 18 years old with a diagnosis of DLBCL between Jan-2011 and Dec-2019 were included in the study. Information on IHC, FISH and karyotype analysis was abstracted from pathology reports or clinical visit notes, where available. Utilization of guideline-recommended diagnostic testing was defined as patients with documentation of a confirmed known result on: 1) expression of MYC, BCL2, BCL6, CD10 or MUM1 protein for IHC; and 2) rearrangement of oncogene MYC, BCL2 or BCL6 by FISH or karyotype analysis for molecular testing. 'Not tested' was defined as patients with no evidence/documentation of diagnostic testing for all of the aforementioned markers. Socioeconomic determinants included age, gender, type of insurance plan, race/ethnicity group and geographic location of residency. We assessed changes in rates of IHC and molecular testing during the study follow-up period using Cochran-Armitage trend tests. To assess the impact of socioeconomic determinants on test utilization, multivariable logistic regression was used to estimate odds ratios (OR) with 95% confidence intervals (CI) for IHC and molecular testing separately, while adjusting for several demographic and clinical characteristics. Results In total 5387 patients with DLBCL were included in the study. Guideline-recommended diagnostic testing rates increased between 2011 and 2019 (66.9 to 85.6% for IHC and 49.5 to 67.0% for molecular testing, both P-values &amp;lt;0.001). In the multivariable-adjusted analysis, no association was found between any socioeconomic determinants and test utilization throughout the study follow-up years for IHC. For molecular testing, however, patients who were older (OR: 0.97, 95% CI: 0.96-0.98), female (OR: 0.77, 95% CI: 0.64-0.93), living in the northeast (OR: 0.75, 95% CI: 0.58-0.97) and without an insurance plan (OR: 0.65, 95% CI: 0.51-0.82) had lower odds of having the test, compared to those who were younger, male, living in the south and with a commercial insurance plan. Regarding racial/ethnic perspective, compared to white patients, Asians were more likely to be associated with having molecular testing (OR: 4.06, 95% CI: 1.81-10.84); no association was found for other racial/ethnic groups. Discussion Although the results of this study showed a significant increase in the adoption of guideline-recommended diagnostic testing for patients with DLBCL in the US between 2011-2019, socioeconomic disparities appeared to still exist in the access to molecular testing. Providing equitable access to diagnostic testing would be a critical parameter for patients' clinical benefit. It should be noted that findings about test utilization need cautious interpretation, as patients with DLBCL might have been tested but not documented in the EHR or might have the test performed at sites outside of the Flatiron Health network. Disclosures Yang: F. Hoffmann-La Roche: Current Employment. Zhang:F. Hoffmann-La Roche: Current Employment. Xiao:F. Hoffmann-La Roche: Current Employment. Hammer:PER Med education: Honoraria; PathEdEx: Current equity holder in private company; Caris Lifesciences: Honoraria; Roche: Consultancy, Honoraria, Research Funding. Prime:F. Hoffmann-La Roche: Current Employment.

Co-Targeting BET Bromodomain Proteins and Aberrant Signaling in AML

AML remains a therapeutic challenge with a high mortality rate, underscoring the need for new biologically based therapies. Somatic alterations that deregulate epigenetic programs and signal transduction pathways frequently coexist in AML. While the former class of alterations is hypothesized to promote a chromatin state that is permissive for AML development and essential for leukemia maintenance, experimental data also suggest that signaling mutations play a central role in driving leukemic growth. Thus, simultaneously targeting the abnormal epigenetic landscape and aberrant signaling is a rational therapeutic approach. We previously used the MEK inhibitor PD0325901 (PD901) to target the Raf/MEK/ERK (MAPK) effector pathway in primary murine AMLs in vivo. Here we report substantial preclinical efficacy of the BET inhibitor PLX51107 in several independent primary AMLs that was further enhanced by PD901. We first exposed five NRAS or KRAS mutant human AML cell lines to varying concentrations of PLX51107 and PD901. These studies revealed potent synergy based on cell proliferation and apoptosis readouts (Figure 1A). We next performed RNA-seq on the NRAS-mutant OCI-AML3 cell line treated with PD901, PLX51107, or the combination. Remarkably, PLX51107 + PD901 cooperated to potently downregulate MYC and its transcriptional targets (Figure 1B). Additionally, AML cells increased the expression of p53 transcriptional targets in response to PLX51107 and CRISPRi-mediated knock-down of TP53 expression abrogated the synergistic effects of PLX51107 + PD901 (Figure 1C). We previously performed retroviral insertional mutagenesis in NrasG12D mice to generate genetically diverse panels of transplantable, primary AMLs. To follow-up on our promising in vitro data, we transplanted 5 of these leukemias into cohorts of recipient mice and treated them with PLX51107 ± PD901. PLX51107 (10 mg/kg/day) showed impressive efficacy that was further enhanced by a modest dose of PD901 (1.5 mg/kg given 4 days per week; Figure 1D). To begin characterizing mechanisms of resistance, we evaluated matched pairs of drug-sensitive ("parental") and relapsed leukemias that emerged during drug treatment. We selected relapsed leukemias that exhibited clonal evolution based on distinct novel retroviral integrations for in depth evaluation. Re-transplanting these AMLs into secondary recipients and re-treating them with PLX51107 ± PD901 confirmed intrinsic drug resistance, which was further validated ex vivo in dose response methylcellulose assays (Figure 1D). Remarkably, several of these resistant leukemias down-regulated basal Myc protein expression, which supports the idea that their transcriptional networks are "rewired" (Figure 1E). We next compared basal and dynamic transcriptional profiles in parental and resistant leukemias by exposing them to PLX51107 ex vivo and then performing RNA-seq analysis. Intriguingly, PLX51107 treatment of parental AMLs resulted in transcriptional changes consistent with myeloid maturation in a dose dependent manner (Figure 1E), suggesting that BET inhibition induces differentiation in these leukemias. By contrast, paired resistant AMLs are relatively "immature" compared to their parental counterparts (Figure 1E), which supports this idea that in vivo treatment with PLX51107 selects for the outgrowth of clones at an earlier progenitor state. These resistant AMLs also upregulate Myc transcriptional targets and unexpectedly downregulate Ras transcriptional targets (Figure 1E). We corroborated these findings by analyzing large pediatric AML transcriptome datasets generated as part of the NCI TARGET initiative (Figure 1F). Taken together, our data support the hypothesis that BET inhibitor and MEK inhibitor combination may be a particularly effective therapeutic approach in AML. Additional preclinical testing in patient derived xenograft models is underway. Figure 1 Disclosures Burgess: Bristol Myers Squibb: Current Employment. Severson:Plexxikon Inc.: Current Employment. Powell:Plexxikon Inc.: Current Employment. Bollag:Plexxikon Inc.: Current Employment.

NCOR2 Mutations Are Associated with IMiD Resistance in Human Myeloma Cells

The immunomodulatory drug thalidomide, and its analogs, lenalidomide, and pomalidomide (IMiDs) have significantly changed the treatment paradigm of multiple myeloma (MM). Despite this progress, IMiD resistance develops in the majority of patients resulting in the development of refractory disease. Cereblon (CRBN), a direct target, has been implicated in IMiD resistance. However, alternate mechanisms of IMiD resistance independent of CRBN remain largely unknown. To understand and study the mechanisms responsible for the development of IMiD resistance, we created lenalidomide-resistant (Len-R) and pomalidomide-resistant (Pom-R) human myeloma MM.1s cell lines, by continuous culture in the presence of lenalidomide or pomalidomide for 3 months. Whole genome sequencing of these 2 resistant cell lines compared with parental MM.1s revealed 172 genes with exonic mutations in both Len-R and Pom-R myeloma cells. Furthermore, a protein-protein interaction (PPI) network was constructed based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The PPI network demonstrated 8 genes that scored a high degree of protein-protein interaction. Among these genes, we identified NCOR2, a corepressor that negatively regulates gene expression, as a downregulated gene in resistant cell lines. To study this further, we created NCOR2 knock out MM.1s cell lines using CRISPR/cas9 gene modification. Our data demonstrates that depletion of NCOR2 confers IMiD resistance independent to CRBN. Interestingly, Len-R, Pom-R and NCOR2 knock out MM.1s showed increased MYC protein expression, which is essential for myeloma cell survival and proliferation. A BET inhibitor, known to disrupt the binding of BRD4 to chromatin, inhibited the proliferation of Len-R and Pom-R and NCOR2 knock out MM.1s by completely suppressing MYC expression. These results indicate that NCOR2 down regulation in IMiD resistant cells induces MYC upregulation which may in part result in IMiD resistance. Our findings reveal a novel molecular mechanism associated with IMiD resistance, independent of CRBN and suggest that NCOR2-MYC pathway may be a new target for IMiD refractory patients. Disclosures Raje: Celgene: Consultancy.

Early Development of a Machine Learning Approach to Quantify MYC Immunohistochemical Staining in Lymphoma

Abstract Newer data suggest that double expression of MYC and BCL2 proteins (DE) evaluated by quantitative immunohistochemistry (qIHC) may be a powerful marker of worse prognosis in diffuse large B cell lymphoma (DLBCL). Testing for DE status, defined as &amp;gt;40% MYC+ and &amp;gt;50% BCL2+ tumor cells, is recommended in the WHO 2016 classification and clinical trials are using DE scoring to assign therapy arms. However, other data suggest that significant variability in manual DE scoring diminishes the predictive value. Error sources include high interobserver variability (IOV) associated with field choice, discrimination of tumor immunoreactivity from adjacent non-neoplastic cells, cell-to-cell variability in staining intensity, crush artifacts and necrosis. Thus, there is a need for standardized, reproducible approaches for DE scoring by qIHC. To address this need, we have begun developing a novel machine-learning approach to analyze IHC digital pathology whole-slide images, focusing initially on MYC IHC. Digital whole-slide images (400x) of 22 DLBCL cases were uploaded to a web-based annotation platform. Using all cases, one annotator created 138 regions of interest (ROIs) containing approximately 200 nucleated cells and representing a variety of tissue types. Eight pathologists were assigned the same 10 ROIs in which to annotate all nuclei from which ground-truth seed nucleus labels (location, classification) were created for a validation set. Nuclei were classified as “tumor-positive”, “tumor-negative”, “non-tumor-positive”, “non-tumor-negative”, or “unknown”. This generated a set of 15,792 annotations with 1974 +/- 272 (Avg+/-STD) labels/annotator. Agglomerative hierarchical clustering afforded the creation of 2299 ground-truth seed locations. A maximum diameter of 3 mm/cluster was set by visual inspection of annotations. Of these seed locations, 1041 (45%) were detected by 8/8 annotators and, on average, 6/8 agreed on class. 302 +/- 72 (Avg+/-STD) “tumor positive” labels per annotator generated 382 seeds locations, 178 (47%) of which were detected by 8/8 annotators, with an average of 7.5/8 agreeing on class. 286 +/- 168 (Avg+/-STD) “tumor-negative” labels per annotator generated 336 seeds, 195 (58%) of which were detected by 8/8 annotators, with an average of 5/8 agreeing on class. Among all classes, the “tumor-positive” label displayed best overall label agreement whereas the “tumor-negative“ label yielded similar localization rate, but lower class agreement. These promising early findings provide a novel basis for quantifying IOV and utilizing multi-observer agreement to create a ground-truth validation set for a supervised machine learning approach to qIHC. Future efforts will make use of these data to optimize the validation set by rationally determining the number of additional annotations required, optimizing the number of annotators per ROI required, devising an adaptive approach to nuclear clustering based on nuclear density, and utilizing the additional 31,422 annotations in hand from all annotators as a robust algorithm training set.

CIP2A regulates MYC translation (via its 5\u2032UTR) in colorectal cancer

BackgroundDeregulated expression of MYC is a driver of colorectal carcinogenesis, suggesting that decreasing MYC expression may have significant therapeutic value. CIP2A is an oncogenic factor that regulates MYC expression. CIP2A is overexpressed in colorectal cancer (CRC), and its expression levels are an independent marker for long-term outcome of CRC. Previous studies suggested that CIP2A controls MYC protein expression on a post-transcriptional level.MethodsTo determine the mechanism by which CIP2A regulates MYC in CRC, we dissected MYC translation and stability dependent on CIP2A in CRC cell lines.ResultsKnockdown of CIP2A reduced MYC protein levels without influencing MYC stability in CRC cell lines. Interfering with proteasomal degradation of MYC by usage of FBXW7-deficient cells or treatment with the proteasome inhibitor MG132 did not rescue the effect of CIP2A depletion on MYC protein levels. Whereas CIP2A knockdown had marginal influence on global protein synthesis, we could demonstrate that, by using different reporter constructs and cells expressing MYC mRNA with or without flanking UTR, CIP2A regulates MYC translation. This interaction is mainly conducted by the MYC 5′UTR.ConclusionsThus, instead of targeting MYC protein stability as reported for other tissue types before, CIP2A specifically regulates MYC mRNA translation in CRC but has only slight effects on global mRNA translation. In conclusion, we propose as novel mechanism that CIP2A regulates MYC on a translational level rather than affecting MYC protein stability in CRC.