TP53 Abnormalities Correlate with Immune Infiltration and Associate

REGULAR ARTICLE

TP53 abnormalities correlate with immune inﬁltration and associate with response to ﬂotetuzumab immunotherapy in AML

Jayakumar Vadakekolathu,1,* Catherine Lai,2,* Stephen Reeder,1 Sarah E. Church,3 Tressa Hood,3 Anbarasu Lourdusamy,4 Michael P. Rettig,5 Ibrahim Aldoss,6 Anjali S. Advani,7 John Godwin,8 Matthew J. Wieduwilt,9 Martha Arellano,10 John Muth,11 Tung On Yau,1 Farhad Ravandi,12 Kendra Sweet,13 Heidi Altmann,14 Gemma A. Foulds,1 Friedrich Stolzel,¨ 14 Jan Moritz Middeke,14 Marilena Ciciarello,15 Antonio Curti,15 Peter J. M. Valk,16 Bob Lowenberg,¨ 16 Ivana Gojo,17 Martin Bornhauser,¨ 14 John F. DiPersio,5 Jan K. Davidson-Moncada,11 Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/20/5011/1779787/advancesadv2020002512.pdf by guest on 27 October 2020 and Sergio Rutella1,18 1John van Geest Cancer Research Centre, School of Science and Technology, Nottingham Trent University, Nottingham, United Kingdom; 2MedStar Georgetown University Hospital’s Lombardi Comprehensive Cancer Center, Washington, DC; 3NanoString Technologies, Inc., Seattle, WA; 4School of Medicine, Biodiscovery Institute, University of Nottingham, Nottingham, United Kingdom; 5Division of Oncology, Department of Internal Medicine, Washington University in St. Louis, St. Louis, MO; 6Department of Hematology and Hematopoietic Cell Transplantation, Gehr Family Center for Leukemia Research, City of Hope, Duarte, CA; 7Leukemia Program, Department of Hematology and Medical Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH; 8Earle A. Chiles Research Institute, Providence Cancer Centre, Portland, OR; 9Moores Cancer Center, University of California San Diego, La Jolla, CA; 10Winship Cancer Institute of Emory University, Atlanta, GA; 11MacroGenics, Inc., Rockville, MD; 12Department of Leukemia, University of Texas MD Anderson Cancer Center, Houston, TX; 13Moffitt Cancer Center, Tampa, FL; 14Department of Internal Medicine I, University Hospital Carl Gustav Carus, Technische Universitat¨ Dresden, Dresden, Germany; 15Institute of Hematology “L. and A. Seragnoli,"´ Department of Hematology and Oncology, University Hospital S. Orsola-Malpighi, Bologna, Italy, 16Department of Hematology, Erasmus Medical Centre, Rotterdam, The Netherlands; 17Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD; and 18Centre for Health, Ageing and Understanding Disease, Nottingham Trent University, Nottingham, United Kingdom

Somatic TP53 mutations and 17p deletions with genomic loss of TP53 occur in 37% to 46% of Key Points acute myeloid leukemia (AML) with adverse-risk cytogenetics and correlate with primary • TP53 mutations corre- induction failure, high risk of relapse, and dismal prognosis. Herein, we aimed to characterize late with enhanced im- the immune landscape of TP53-mutated AML and determine whether TP53 abnormalities mune infiltration and identify a patient subgroup that may benefit from immunotherapy with flotetuzumab, an high levels of actionable investigational CD123 3 CD3 bispecificdual-affinity retargeting antibody (DART) molecule. immune checkpoints in The NanoString PanCancer IO360 assay was used to profile 64 diagnostic bone marrow (BM) AML. samples from patients with TP53-mutated (n 5 42) and TP53-wild-type (TP53-WT) AML (n 5 22) • TP53 mutations asso- and 45 BM samples from patients who received flotetuzumab for relapsed/refractory (R/R) ciate with complete AML (15 cases with TP53 mutations and/or 17p deletion). The comparison between TP53- responses to flotetuzu- mutated and TP53-WT primary BM samples showed higher expression of IFNG, FOXP3, mab, an investigational immune checkpoints, markers of immune senescence, and phosphatidylinositol 3-kinase-Akt immunotherapy, in R/R AML. and NF-kB signaling intermediates in the former cohort and allowed the discovery of a 34- geneimmuneclassifier prognostic for survival in independent validation series. Finally, 7 out of 15 patients (47%) with R/R AML and TP53 abnormalities showed complete responses to flotetuzumab (,5% BM blasts) on the CP-MGD006-01 clinical trial (NCT #02152956) and had significantly higher tumor inflammation signature, FOXP3, CD8,inflammatory chemokine, and PD1 gene expression scores at baseline compared with nonresponders. Patients with TP53 abnormalities who achieved a complete response experienced prolonged survival (median, 10.3 months; range, 3.3-21.3 months). These results encourage further study of flotetuzumab immunotherapy in patients with TP53-mutated AML.

Submitted 4 June 2020; accepted 24 August 2020; published online 15 October based upon data generated by the TCGA Research Network (https://www.cancer. 2020. DOI 10.1182/bloodadvances.2020002512. gov/tcga). *J.V. and C.L. contributed equally to this study. The full-text version of this article contains a data supplement. The data reported in this article have been deposited in the Gene Expression Omnibus © 2020 by The American Society of Hematology database (accession number GSE146204). The results shown in this paper are in part

27 OCTOBER 2020 x VOLUME 4, NUMBER 20 5011 Introduction Table 1. Patient series Wet laboratory In silico SAL Bologna TCGA* Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease. The patients with the poorest outcomes No. of patients 40 24 147 are those with refractory disease, including individuals with primary Males/females, n 26/14 17/7 81/66 1 induction failure (PIF) who fail initial induction attempts. Complex Age, n karyotypes, including deletions affecting chromosome arm 17p, 0-14 y 0 0 0 frequently coexist with TP53 mutations and define a “very unfavor- 15-39 y 2 3 27 able” subgroup of AML with a 5-year relapse-free survival (RFS) and overall survival (OS) of 0%.2-4 Somatic TP53 mutations and 40-59 y 13 12 45 deletions of 17p, to which TP53 is mapped, occur in 8% to 10% .60 y 25 9 75 Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/20/5011/1779787/advancesadv2020002512.pdf by guest on 27 October 2020 of de novo AML5-7 and in up to 37% to 46% of patients with adverse- WBC count at presentation, median 10.55 (0.8- 45 (1.5- 20 (1-297) 3 3 risk cytogenetics and treatment-related myeloid neoplasms.2,8 Further- (range), 10 /mL 218.5) 153) more, patients with mutated TP53 and/or 17p deletion tend to be older Percentage of BM blasts, median (range) 63.7 (30-90) 16.5 (0.4- 72 (11-99) and have low performance status, and therefore, only a few of them are 57) candidates for allogeneic hematopoietic stem cell transplantation Cytogenetic risk group, n (%) (HSCT), which offers the highest curative potential.9 Response rates ELN favorable 0 (0) 6 (26.1) 17 (12) to standard-of-care (SOC) cytarabine-based induction chemotherapy ELN intermediate 0 (0) 7 (30.4) 96 (65) in patients with TP53-mutated AML range between 14% and 42%, ELN adverse 40 (100) 5 (21.7) 32 (22) with a median OS of 2 to 12 months.2,5 In the relapsed and refractory NA 0 (0) 5 (21.7) 2 (1) (R/R) setting, where TP53 mutations are highly prevalent, response TP53 rates to standard salvage cytotoxic regimens are ,20%.2,10,11 status Mutated 40 2 14 Emerging evidence implicates mutant TP53, in addition to its well- WT 0 22 — characterized function as a tumor suppressor, in activating genes —— involved in immune responses and inflammation, including chemo- Not tested/not available 133 kines, cytokines, and extracellular matrix modulators.12 A recent Induction chemotherapy analysis of The Cancer Genome Atlas (TCGA) transcriptomic data 7 1 3 5 2 113 from 10 000 nonhematologic tumors has indicated that TP53 Fludarabine based — 8 — mutations correlate with increased leukocyte infiltration across 30 Daunorubicin 1 cytarabine 21 0 — diverse cancer types and are enriched in the C1 (wound healing) MAV 12 5 — and C2 (interferon-g [IFN-g] dominant) immune subtypes.13 1 Importantly, higher proportions of PD-L1–expressing CD8 HMAs — 314 T cells, higher tumor mutational burden, and increased expression Lenalidomide 1 — 9 of T-cell effector genes and IFN-g–related genes associate with Other 1 6 11 favorable responses to pembrolizumab immunotherapy in patients Cohort-wide OS (mo from diagnosis), 5.06 (0.03- 16.5 (0.3- 15.5 (0.1- 14 with TP53-mutated lung cancer. median (range) 158.3) 57) 118.1) We have recently identified microenvironmental immune gene sets SAL, Studien-Allianz Leukamie;¨ ELN, European Leukemia-Net; HMAs, hypomethylating – – agents; MAV, mitoxantrone, cytarabine, and etoposide; WBC, white blood cell. that capture elements of both type I and IFN-g driven biology and *Cases of newly diagnosed nonpromyelocytic AML with RNA-sequencing data and stratify newly diagnosed AML into an immune-infiltrated and an clinical annotation. immune-depleted subtype.15 Our immune classifier increased the accuracy of survival prediction in patients receiving cytarabine- based induction chemotherapy beyond the current capabilities of individual molecular markers. Herein, we aimed to investigate newly diagnosed AML treated with curative intent (Bologna whether TP53 mutations shape the immune landscape of AML and cohort; 2 cases with mutated TP53). The third wet-laboratory whether they identify patients that derive benefit from flotetuzumab, cohort consisted of 45 primary BM samples from patients with an investigational CD123 3 CD3 bispecific dual-affinity retargeting PIF,early-relapseAML(durationofcompleteremission1[CR1] antibody (DART) molecule.16 ,6 months), and late-relapse AML (duration of CR1 $6 months) treated with flotetuzumab at the recommended phase Materials and methods 2 dose (500 ng/kg per day) on the CP-MGD006-01 clinical trial (NCT #02152956). Patients with prior HSCT were ineligible. Patient demographics and study approval Fifteen patients from the flotetuzumab cohort harbored TP53 Patient and disease characteristics as well as induction treatment mutations or 17p deletions with genomic loss of TP53. Patients regimens are summarized in Table 1. TP53 mutational status is received a lead-in dose of flotetuzumab during week 1, followed by detailed in supplemental Tables 1 and 2. The first wet-laboratory 500 ng/kg per day during weeks 2 to 4 of cycle 1 and a 4-day on/ cohort consisted of 40 primary bone marrow (BM) samples 3-day off schedule for cycle 2 and beyond. Disease status was from patients with newly diagnosed, TP53-mutated AML treated assessed by modified International Working Group criteria.17 with curative intent (SAL cohort). The second wet-laboratory Specifically, complete responses were defined as either CR, CR cohort included 24 primary BM samples from patients with with partial hematological recovery (CRh), CR with incomplete

5012 VADAKEKOLATHU et al 27 OCTOBER 2020 x VOLUME 4, NUMBER 20 A B

Molecular lesion 6 Molecular lesion B7–H3 FLT3–ITD Apoptosis TGF–beta 4 NPM1+FLT3–ITDneg MMR.loss Other 40 0.6 APM.loss 2 Mast cells RUNX1 PD1 P 0.0001 P 0.0001 CTLA4 0 TP53 Treg B cells Th1 cells –2 30 Cytotoxicity 0.4 Cytotoxic cells NK CD56dim cells Exhausted CD8 TIGIT T cells Lymphoid cells 20 CD8 T cells NK cells 0.2 Stroma Endothelial.cells Inflammatory.chemokines MHC2 Immunoproteasome 10 APM CD45 0.0 Macrophages

Myeloid cells Number of mutations Neutrophils IL10 0 Fraction of genome altered Fraction

PDL1 Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/20/5011/1779787/advancesadv2020002512.pdf by guest on 27 October 2020 PDL2 TIS IFN.downstream -0.2 IFN–gamma DC IDO1 TP53-mut. TP53 wt TP53-mut. Other Glycolytic.activity Hypoxia Myeloid.inflammation ARG1 Proliferation MAGEs NOS2

Immune-infiltrated Immune-depleted (n=14 with TP53-mut.) (n=0 with TP53 mut.)

C D 9 8 9 9 8 P = 0.0138 P = 0.0252 P = 0.0019 KW = 53.03 KW = 27.96 P 0.0001 P 0.0001 7 6 6 6 6 4 5 score 4 3

2 TIS score IFN- TIS score 3 3 2 0 0 1 Inflammatory score chemokine 0 Inflammatory score chemokine 0 -3

Mut. Mut. Mut. Mut. TP53 CK with TP53 CK with TP53 CK with NPM1 ) NPM1 ) RUNX1 RUNX1 mut wt TP53 mut wt TP53 mut wt TP53 NPM1 TP53 NPM1 TP53 Mut. Mut. FLT3-ITDMut. FLT3-ITDMut. CHIP-defining CHIP-defining w/o (ASXL1,TET2,DNMT3A w/o (ASXL1,TET2,DNMT3A

10 8 12 P = 0.0056 KW = 20.9 KW = 40.1 6 P 0.0003 10 P 0.0001 8 4 8 score

6 2 6 IFN- Lymphoid score Lymphoid 0 4 score Lymphoid 4

-2 2 2

Mut. Mut. Mut. Mut. TP53 CK with NPM1 ) NPM1 ) RUNX1 RUNX1 mut wt TP53 NPM1 TP53 NPM1 TP53 Mut. Mut. FLT3-ITDMut. FLT3-ITDMut. CHIP-defining CHIP-defining w/o (ASXL1,TET2,DNMT3A w/o (ASXL1,TET2,DNMT3A

P = 0.0002 12 P 0.0001 12 9 P = 0.0002 ) ) ) 2 2 9 2 9 6 6 6 mRNA (log mRNA (log mRNA (log

3 3 TIGIT PD-L1 3 FoxP3

0 0 0 TP53-mut. Other TP53-mut. Other TP53-mut. Other

Figure 1. TP53 mutations correlate with an immune-infiltrated TME in TCGA-AML. (A) Heatmap of immune-cell-type–specific scores and biological activity scores in TCGA-AML cases with information on prognostic molecular lesions (n 5 118; unsupervised hierarchical clustering; Euclidean distance; complete linkage). ClustVis, an online tool for clustering of multivariate data, was used for data analysis and visualization.47 The optimal number of clusters was defined through silhouette scoring using an open- source machine learning toolkit (Orange3, version 3.25.0). FLT3-ITD, fms-like tyrosine kinase 3 internal tandem duplication; NPM1, nucleophosmin-1. ELN intermediate cases were further subclassified into molecular low-risk cases (NPM1 mutations without FLT3-ITD) and molecular high-risk cases (NPM1 WT with FLT3-ITD).22 (B) Fraction of genome altered in TCGA-AML cases with TP53 mutations (n 5 14) and other prognostic molecular lesions (n 5 104). Bars denote median values. Data were retrieved through cBioPortal and were compared using the Mann-Whitney U test for unpaired determinations. (C) Box plots showing immune signature scores in TCGA-AML cases with TP53 mutations (n 5 14) and other prognostic molecular lesions (n 5 19 with NPM1 mutations; n 5 22 with RUNX1 mutations; n 5 48 with FLT3-ITD without NPM1 mutations; n 5 15 with CHIP-defining mutations). Data were compared using the Kruskal-Wallis test for unpaired determinations with Bonferroni’s correction for multiple comparisons. (D) TIS, inflammatory chemokine, IFN-g, and lymphoid signature scores in TCGA cases with TP53 mutations with (n 5 9 cases) or without a CK (n 5 13 cases)

27 OCTOBER 2020 x VOLUME 4, NUMBER 20 TP53 MUTATIONS AND IMMUNOTHERAPY RESPONSE IN AML 5013 hematological recovery (CRi), or morphological leukemia-free deletion) were present in 14 patients (lollipop plot in supplemental state (MLFS) at the end of cycle 1. Other responses were defined Figure 1A and supplemental Table 2). TP53-mutated cases harboring as either partial response (.50% decrease in BM blasts from comutations in genes with known prognostic relevance in AML, such baseline or decrease to 5% to 25% BM blasts) or other benefit as DNMT3A and TET2,23-25 were excluded from the analysis (.30% reduction of BM blasts from baseline) at the end of cycle (supplemental Figure 1B). As expected, median OS from diagnosis 1. Human studies were approved by the institutional review boards was 4.5 months in TCGA cases with mutated TP53 compared with at SAL (Germany) and the University of Bologna (Italy), and by the 18.5 months in patients with other prognostic molecular lesions institutional review boards of the centers participating to the (hazard ratio [HR, 3.43; P , .0001; supplemental Figure 1C). flotetuzumab immunotherapy clinical trial. Written informed consent was received from all participants prior to inclusion in the study. As shown in Figure 1A-B, TP53-mutated AML cases showed higher immune infiltration, a higher number of mutations, and a higher Details on immune gene expression profiling, in silico data sources, fraction of genome altered (fraction of copy-number–altered chromo- gene set enrichment analysis, in vitro propagation of AML cell lines some regions out of measured regions), consistent with the role of Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/20/5011/1779787/advancesadv2020002512.pdf by guest on 27 October 2020 and flow cytometry-based assays are provided in the supplemental mutant TP53 in promoting genomic instability,26 compared with Appendix and in previous publications.15,18 patients harboring other high-risk molecular features (NPM1 WT with FLT3-ITD) and patients with low or intermediate risk. The TIS score Statistical analyses was significantly higher in TP53-mutated cases relative to cases with molecular lesions associated with favorable clinical outcomes (NPM1 Descriptive statistics included calculation of mean, median, mutations without FLT3-ITD) and cases with clonal hematopoiesis of standard deviation, and proportions to summarize study out- indeterminate potential (CHIP)–defining mutations in ASXL1, TET2, comes. Comparisons were performed with the Mann-Whitney and DNMT3A (P , .0001; Figure 1C). Overall, the higher IFN-g U test for paired or unpaired data (2 sided), as appropriate, or signaling, inflammatory chemokine, and lymphoid scores in patients with the analysis of variance with correction for multiple comparisons. with TP53 mutations suggested a higher degree of immune infiltration A2-tailedP , .05 was considered to reflect statistically significant and the activation of IFN-g–related signaling pathways (Figure 1C). differences. The log-rank (Mantel-Cox) test was used to compare TP53 alterations commonly occur in patients with complex karyotype survival distributions. OS was computed from the date of diagnosis to (CK) AML.4 As shown in Figure 1D, the TIS, IFN-g signaling, the date of death. RFS was measured from the date of first CR to the inflammatory chemokine, and lymphoid signature scores were higher date of relapse or death. Subjects lost to follow-up were censored at in TP53-mutated AML than CK AML with WT TP53. This observation their date of last known contact. IBM SPSS Statistics (version 24) suggests that changes in immune gene expression are unlikely to be and GraphPad Prism (version 8) were used for statistical analyses. attributable to chromosome gains and losses. Results The expression of actionable immune checkpoints (PD-L1 and TP53 mutational status correlates with immune TIGIT) and genes associated with a highly immune-suppressed TME, such as the regulatory T (Treg) cell transcription factor infiltration in TCGA-AML cases FOXP3,washigherinTP53-mutated cases (Figure 1E; P We first asked whether the expression of known AML drivers, values adjusted for multiple testing). Gene set enrichment including TP53, correlates with the immune composition and analysis using published gene sets from landmark studies 1 functional orientation of the BM tumor microenvironment (TME). To characterizing CD8 T-cell dysfunction in AML (supplemental address this hypothesis, we retrieved RNA-sequencing data with Table 3),27 as well as curated gene sets from the Molecular cytogenetic and clinical annotation, including RFS and OS, from Signature Database, indicated functional enrichment of an adult patients with newly diagnosed, nonpromyelocytic AML immune senescence–related 8-gene set in TCGA-AML cases profiled in the TCGA project (n 5 147 cases available through with TP53 mutations (supplemental Figure 2A-B). In contrast, cBioPortal for Cancer Genomics; n 5 118 cases with information no enrichment was observed in TP53-mutated cases when on prognostic molecular lesions). Signature scores that identify probing gene sets defining T-cell exhaustion (supplemental immune subtypes of AML with poor sensitivity to induction Figure 2A). The enrichment of TP53-mutated BM samples with 1 chemotherapy and worse clinical outcomes were derived from CD8 T cells, Treg cells, and other innate and adaptive immune messenger RNA (mRNA) expression, as previously published cell types was validated using CIBERSORT, a deconvolution (supplemental Table 1),15,19 and included the tumor inflamma- algorithm that allows the enumeration of 22 functionally defined tion signature (TIS), an established predictor of response to hematopoietic cell subsets from gene expression profiles, including immune checkpoint blockade in solid tumors.20,21 ELN in- T-cell subsets, naive and memory B cells, plasma cells, natural killer termediate cases with information on NPM1 mutational status cells, and myeloid-cell subsets (supplemental Figure 2C).28 The and FLT3-ITD were further subclassified into molecular low increased T-cell frequency in TP53-mutated AML (n 5 3) compared risk (NPM1 mutations without FLT3-ITD) and molecular high with TP53-WT AML (n 5 13) and BM samples from healthy donors risk (NPM1 wild-type [WT] with FLT3-ITD).22 TP53 mutations (8 (n 5 4) was also confirmed using publicly available single-cell RNA- missense, 3 frameshift, 3 splice site, 1 nonsense, and 1 homozygous sequencing datasets (GSE116256; supplemental Figure 3).29

Figure 1. (continued) for whom immune gene signature scores could be computed. Data were compared using the Mann-Whitney U test for unpaired determinations. (E) Expression of FOXP3 and immune checkpoints PD-L1 and TIGIT in TCGA-AML cases with TP53 mutations (n 5 14) and other prognostic molecular lesions (n 5 104). Bars denote median values. Data were compared using the Mann-Whitney U test for unpaired determinations. ND, not determined.

5014 VADAKEKOLATHU et al 27 OCTOBER 2020 x VOLUME 4, NUMBER 20 A B

TP53 status Mutated WT

84% TP53 missense 10

12% TP53 no missense

4% N.A. 5

0 PC2 (9.6%) C –5

TP53 status Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/20/5011/1779787/advancesadv2020002512.pdf by guest on 27 October 2020 TP53 status Mast cells 4 Mutated ARG1 –10 Proliferation WT JAK/STAT loss 2 APM loss MHC2 Immunoproteasome 0 –10 –5 0 5 10 TGF–beta Neutrophils Myeloid cells –2 PC1 (41.5%) Macrophages CTLA4 CD8 T cells –4 Lymphoid cells NK cells T–Cells D Th1 cells CTL Cytotoxicity TIGIT CD45 TP53 status B cells TP53 status 3 MMR loss Cell proliferation Mutated Apoptosis DNA damage repair 2 WT Endothelial cells Autophagy 1 Stroma Hedgehog signaling IL10 Wnt signaling 0 Myeloid inflammation Costimulatory signaling –1 Inflammatory chemokines Lymphoid compartment –2 Glycolytic activity Interferon signaling Hypoxia Cytotoxicity –3 Exhausted CD8 T cells NF–kB signaling IFN downstream JAK–STAT signaling TIS Cytokine and chemokine signaling IDO1 Metabolic stress IFN–gamma MAPK CD56dim NK cells TGF–beta signaling PD1 NOS2 Hypoxia DC PI3K–Akt signaling MAGEs Notch signaling APM Antigen presentation Treg cells Immune cell adhesion and migration PDL1 Apoptosis PDL2 Myeloid compartment B7–H3 Matrix remodeling and metastasis Angiogenesis Epigenetic regulation Immune high Immune intermediate Immune low (20/23 TP53-mut.) (21/28 TP53-mut.) (1/13 TP53-mut.)

E F 10 10 10 14 9 P=0.048 P=0.045 P=0.016 16 P<0.0001 P<0.0001 P=0.0013 12 ) ) 2 ) 2 5 5 5 2 10 12 6 8 0 0 0 8 mRNA (log mRNA (log 6 mRNA (log B signaling score -

CD8A

DNA damage score 4 PD-L1 -5 -5 -5 IFN NF- 4 JAK/STAT signaling score 2

-10 -10 -10 0 0 0 No mis. Mis. WT No mis. Mis. WT No mis. Mis. WT TP53-WT TP53-mut TP53-WT TP53-mut TP53-WT TP53-mut TP53 status TP53 status TP53 status

6 4 15 9 12 15 P<0.0001 P=0.0004 P=0.0013 P<0.0001 P<0.0001 P=0.042 4 ) 10 ) 9 ) 2 2 2 2 2 6 10 5 6 0 0 mRNA (log mRNA (log mRNA (log 0 -2 3 3 5

LAG3 FoxP3 GZMB Wnt signaling score Wnt -2 PI3K-Akt signaling score

Hedgehog signaling score -5 -4 0

-6 -4 -10 0 0 No mis. Mis. WT No mis. Mis. WT No mis. Mis. WT TP53-WT TP53-mut TP53-WT TP53-mut TP53-WT TP53-mut TP53 status TP53 status TP53 status

Figure 2. TP53-related cancer pathways and expression of actionable immune checkpoints in patients with TP53-mutated AML (SAL and Bologna cohorts). (A) TP53 mutations were categorized as missense or no missense (frameshift, splice site and nonsense) using the IARC TP53 database (http://p53.iarc.fr/) and based on prior knowledge.30,31 (B) Principal-component (PC) analysis of 770 immune genes (IO 360 panel) in patients with TP53-mutated (n 5 42) and TP53WT AML (n 5 22). Points are colored by TP53 mutational status (mutated, red; WT, blue). ClustVis was used for data analysis and visualization. (C) Heatmap of immune-cell-type–specific and biological activity scores in patients with TP53-mutated and TP53-WT AML (unsupervised hierarchical clustering; Euclidean distance; complete linkage). The number of TP53-mutated cases in each immune cluster (high, intermediate, and low) is indicated. ClustVis, an online tool for clustering of multivariate data, was used for data analysis and visualization. (D) Heatmap of cancer pathway scores in patients with TP53-mutated and TP53-WT AML (unsupervised hierarchical clustering; Euclidean distance; complete linkage). (E) Cancer pathway scores in patients with TP53-mutated and TP53-WT AML. Bars denote median values. Data were compared using the Kruskal-Wallis test for unpaired determinations. (F) Box plots summarizing the expression of negative immune checkpoints and immune genes related to T-cell infiltration, regulatory T cells, and cytolytic activity

27 OCTOBER 2020 x VOLUME 4, NUMBER 20 TP53 MUTATIONS AND IMMUNOTHERAPY RESPONSE IN AML 5015 A B

VTCN1

20 TP53 status TP53 status FCAR 4 NS CXCR2 Mutated PRF1 2 WT Log FC GIMAP6 2 CD79A CXCL8 0 P value ITGB3 15 OASL –2 P value and Log FC THBS1 2 IL33 RIPK2 GBP1 P CXCL2 CD79B FAM30A PTGER4 RIPK2 10 THBD DUSP5 MS4A1 CCL2

adjusted value SMAD5 PTGER4 CX3CR1 10 CD2 CXCL2 DUSP5 CXCL1 MARCO BBC3 CCL3/L1 ITGB3 NECTIN2 CCL3/L1 THBD GBP1 NT5E MYCT1 IL33 -Log CD19 OASL CCR2 TNFAIP3 CSF1 GIMAP6CCR2 IFNG 5 CXCR2 FCAR CXCL8 MS4A1 IFNG Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/20/5011/1779787/advancesadv2020002512.pdf by guest on 27 October 2020 PRF1 CD79A AREG IL6 MYCT1 GIMAP4 CDKN2ACXCL3 CXCL1 CD19 CD79B CX3CR1 FCN1 THBS1 FAM30A CCL2 CSF1 TNF VTCN1 CD2 MARCO 0 IL6 NT5E BBC3 –5 –4 –3 –2 –1 0 1 2 3

Log2 fold change Up in TP53-wt AML Up in TP53-mut. AML (n=22 cases) (n=42 cases)

THBS1 IL34

CSF1R TCGA-AML FGG THBD 50 FGA CSF1 Query genes altered (n=93) F2 P=0.044 OASL Query genes not altered (n=54) MX1 40 SERPINC1 IL1RL1 SERPIND1 IL33 30

IL6 IFNG P=0.01 FCAR 20

IL6R RIPK2

Mutation frequency (%) 10

PTGER4 CXCL1 DUSP5 CCL2 0

MYCT1 ITD CXCL8 TP53

CXCL2 NPM1 RUNX1 FLT3- BBC3 DNMT3A

Figure 3. Identification of a TP53-related immune gene set in patients with TP53-mutated AML (SAL and Bologna cohorts). (A) Volcano plot (R package version

1.4.0) showing DE genes (P threshold 5 .01; log2 fold change $1.5) between patients with TP53-mutated (n 5 42) and TP53-WT AML (n 5 22). B) Heatmap displaying the

34 genes with the greatest differential expression between patients with TP53-mutated and TP53-WT AML (P threshold 5 .01; log2 fold change $1.5). ClustVis, an online tool for clustering of multivariate data, was used for data analysis and visualization. (C) Analysis of functional protein association networks using STRING (https://string-db.org/). Top 10 molecules interacting with DE genes between TP53-mutated and TP53-WT AML are shown together with their predicted mode of action (highest-confidence interaction scores .0.900). Network nodes (query proteins) represent proteins produced by a single protein-coding gene locus. White nodes represent second shells of interactors. Empty and filled nodes indicate proteins of unknown or partially known 3-dimensional structure, respectively. Edges represent protein-protein associations. Line shapes denote predicted modes of action. (D) Correlation between abnormalities of genes in the TP53 immune classifier and prognostic molecular lesions, including TP53 mutations, in TCGA-AML cases. Data were retrieved and analyzed using cBioPortal for Cancer Genomics (http://www.cbioportal.org/) and were compared using the Fisher’s exact test. NS, not significant.

Primary BM samples from patients with supplemental Table 4 and were inferred using the IARC TP53 30,31 TP53-mutated AML express inflammatory and database (http://p53.iarc.fr/) and prior knowledge. These IFN-related gene sets dataareinagreementwiththe reported frequency of TP53 missense mutations (83.7%) in the German-Austrian AML We next compared immune gene expression profiles between bulk series.32 Our set of predefined immune-cell-type–specific BM specimens from patients with TP53-mutated (n 5 42) and TP53- scores and biological activity scores distinguished patients WT AML (n 5 22). The predicted functional consequences with TP53-mutated AML from individuals with TP53-WT AML, of TP53 mutations (84% missense; Figure 2A) are listed in as highlighted by data dimensionality reduction using principal-

Figure 2. (continued) in patients with TP53-mutated and TP53-WT AML. Bars denote median values. Data were compared using the Mann-Whitney U test for unpaired determinations. NA, not available; PI3K, phosphatidylinositol 3-kinase.

5016 VADAKEKOLATHU et al 27 OCTOBER 2020 x VOLUME 4, NUMBER 20 A TCGA-AML B

10 12 P = 0.0008 P = 0.0039 Classifier Classifier 6 B7–H3 Altered Macrophages Not altered 9 Myeloid cells 4 Neutrophils 5 IL10 MHC2 2 score

Immunoproteasome 6 APM PDL2 0 TIGIT score TIS IFN- IFN.downstream 0 IFN–gamma –2 3 DC IDO1 Mast cells B cells TIGIT 0 T cells Lymphoid cells CD8 T cells Altered Not Altered Not Exhausted CD8 altered NK cells altered Th1 cells Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/20/5011/1779787/advancesadv2020002512.pdf by guest on 27 October 2020 Cytotoxicity Cytotoxic cells NK CD56dim cells CTLA4 12 Treg P = 0.001 P = 0.0003 PDL1 9 Stroma Endothelial.cells Inflammatory.chemokines 9 CD45 MAGEs 6 Glycolytic.activity Hypoxia Myleoid.inflam 6 ARG1 Proliferation 3 NOS2 CD8 score PD1 Apoptosis 3 TGF–beta MMR.loss 0 APM.loss Inflammatory chemokines 0 Altered Not Altered Not C altered altered

100 Altered (median RFS=11.4 mo.) 100 Altered (median OS=11.4 mo.) Not altered (median RFS=24.1 mo.) Not altered (median OS=27.0 mo.) 10 P = 0.016 75 75 P = 0.0027 10 8 50 50 7 6 Percent survivalPercent Percent survivalPercent 25 P = 0.0068 25 P = 0.0009 4 4 2 2 Log rank =7.32; HR=1.86 (95%CI 1.12-3.1) Log rank =10.94; HR=1.88 (95%CI 1.24-2.85) PD-L1 score 0 0 1 IFN downstream score 2 0 24 48 72 96 120 144 0 24 48 72 96 120 144 Relapse-free survival time (months) Overall survival time (months) 0 -2 Altered Not Altered Not altered altered

Figure 4. Upregulated genes in the TP53 immune gene classifier correlate with immune infiltration in TCGA-AML cases. (A) Heatmap of immune-cell-type– specific scores and biological activity scores (unsupervised hierarchical clustering; Euclidean distance; complete linkage) in TCGA-AML cases with (n 5 93)orwithout (n 5 54) abnormalities in the DE genes (TP53 immune classifier) between patients with TP53-mutated (n 5 42) and TP53-WT AML (n 5 22). Abnormalities were defined as mRNA upregulation, gene amplification, deep deletion, and missense mutations relative to the gene’s expression distribution in all profiled AML samples. Abnormalities in only 1 gene used in the cBioPortal query (by default, nonsynonymous mutations, fusions, amplifications, and deep deletions) were sufficient to define that particular patient sample as “altered." (B) Expression of IFN-g signaling molecules, immune checkpoints, and markers of T-cell infiltration in TCGA-AML cases with or without abnormalities in the TP53 classifier genes. Bars denote median values. (C) Abnormalities in the 18 upregulated immune genes in the TP53 classifier in pretreatment BM samples from TCGA-AML cases. Data were retrieved, analyzed, and visualized using cBioPortal.34 The Kaplan-Meier method was used to generate survival curves, which were compared using a log-rank test. component analysis (Figure 2B). The frequency of TP53- (Figure 2F), a molecule recently associated with features of 1 mutated cases in patients with high, intermediate, and low exhaustion and senescence in AML-infiltrating CD8 T cells27 immune infiltration was 87% (n 5 20/23), 75% (n 5 21/28), and with worse clinical outcomes.15 and 8% (n 5 1/13), respectively (Figure 2C). We also computed scores that capture frequently dysregulated signal- We next analyzed the immune transcriptomic profile at the gene ing pathways in cancer using predefined sets of relevant genes. level, and we identified a set of 34 differentially expressed (DE) AsshowninFigure2D,TP53-mutated cases expressed higher immune genes at a false discovery rate ,0.01 between patients amounts of NF-kB, JAK/STAT, and phosphatidylinositol 3-kinase with TP53-mutated and TP53-WT AML (Figure 3A-B; supple- (PI3K)-Akt signaling molecules relative to BM samples from mental Table 5). The TP53 immune signature genes have not patients with TP53-WT AML. In contrast, DNA damage repair been previously implicated in the TP53 pathway (supplemental genes as well as Hedgehog and Wnt signaling pathway genes Figure 4A). Neutrophil chemoattractants (proinflammatory CXCL1, were upregulated in TP53-WT compared with TP53-mutated CXCL2,andCXCL8 or IL8) and IFN-inducible molecules AML (Figure 2E). These findings are congruent with previous such as CCL2, IL33, IL6, OASL,andRIPK2 were more studies showing that TP53 is a suppressor of canonical Wnt highly expressed in TP53-mutated compared with TP53- signaling in solid tumors.33 TP53-mutated AML also expressed WT patients. The DE genes exhibited enrichment of Gene IFNG, FOXP3, PD-L1, LAG3, CD8A, PTGER4,andGZMB Ontology and Kyoto Encyclopedia of Genes and Genomes

27 OCTOBER 2020 x VOLUME 4, NUMBER 20 TP53 MUTATIONS AND IMMUNOTHERAPY RESPONSE IN AML 5017 A B

CD34–mRNA NS

LYN–mRNA 6 RAC1–mRNA Log FC STAT6–mRNA SYK–mRNA 2 CBL–mRNA 1 PIM1–mRNA CCND3–mRNA p53–protein IKKb–protein CBL–mRNA P value CSF3R–mRNA 0.5 IRF1–mRNA DNMT3A–mRNA FYN–mRNA SET–mRNA STAT6–mRNA P value and Log FC STAT1–mRNA 2 FOXO1–mRNA 0 TP53–mRNA KAT6A–mRNA STAT5a– protein DDIT3–mRNA –0.5 IKZF2–mRNA IFI16–mRNA KIT–mRNA RHOA–mRNA CDKN2A–mRNA DNMT3A–mRNA CCND2–mRNA STAT5A–mRNA –1 PTPRC–mRNA CSF3R–mRNA CD79A–mRNA IDH1–mRNA CHEK1–mRNA –1.5 4 HOXA9–mRNA S6–protein JAK2–mRNA IL2RA–mRNA Ik–Ba–protein P KIT mRNA p53–protein EIF4E–mRNA EIF4E–mRNA BCL3–mRNA CDKN2A–mRNA MYCN–mRNA NLRC5–mRNA HGF–mRNA SOCS3–mRNA LCK–mRNA RARA–mRNA DDIT3–mRNA FYN–mRNA MEIS1–mRNA HGF–mRNA CIITA–mRNA CHEK2–mRNA SOCS1–mRNA IDH1–mRNA CCND2–mRNA NF–kB–protein Alpha–Tubulin–protein NLRC5–mRNA MYCN–mRNA RARA–mRNA adjusted value MEIS1–mRNA LCK–mRNA RHOA–mRNA 10 OSM–mRNA KAT6A–mRNA FOXO1–mRNA CHEK1–mRNA TP53–mRNA CD34–mRNA CD79A–mRNA IKKb–protein JAK2–mRNA RAC1–mRNA 2 IL2RA–mRNA –Log SET–mRNA CCND3–mRNA

IFI16–mRNA Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/20/5011/1779787/advancesadv2020002512.pdf by guest on 27 October 2020 IKZF2–mRNA IRF1–mRNA STAT1–mRNA SYK–mRNA PIM1–mRNA LYN–mRNA OSM–mRNA HOXA9–mRNA NF–kB–protein Ik–Ba–protein BCL3–mRNA 0 CIITA–mRNA KG-1 AML Kasumi-1 AML –7.5 –5.0 –2.5 0.0 2.5 5.0 7.5 10.0 (TP53 LOF) (TP53 GOF) Log2 fold change Up in KG-1 AML Up in Kasumi-1 AML (TP53 LOF) (TP53 GOF)

CD TCGA-AML

1.0 Low 34 Signatures Group 1.0 Low 10 Signatures Group High 34 Signatures Group High 10 Signatures Group Cell line Logrank p=0.048 Logrank p=0.92 Cell line 1 TCR Signaling Kasumi1 0.8 HR(high)=1.7 0.8 HR(high)=0.97 KG1 BCR Signaling 0.5 p(HR)=0.053 p(HR)=0.92 Immunoregulation n(high)=53 n(high)=53 NF–kB 0 Cell Cycle n(low)=53 n(low)=53 Cytokine Signaling –0.5 0.6 0.6 MYC JAK–STAT –1 PI3K Apoptosis & DNA Damage –1.5 Epigenetics and Transcription 0.4 0.4 Driver Gene MAPK Percent survivalPercent survivalPercent

0.2 0.2

0.0 KG-1 AML signature 0.0 Kasumi-1 AML signature

020406080 020406080 Months Months

Figure 5. Integrated mRNA and protein profiling of AML cells lines with missense and truncating TP53 mutations. (A) Volcano plot (R package version 1.4.0) showing DE mRNA species and proteins between AML cell lines with missense (Kasumi-1 cells; p.R248Q; Broad Institute Cancer Cell Line Encyclopedia) and truncating (splice site) mutations of TP53 (KG-1 cells).30 (B) Heatmap of the top DE mRNA species and proteins between KG-1 AML and Kasumi-1 AML (unsupervised hierarchical clustering; Euclidean distance; complete linkage). ClustVis, an online tool for clustering of multivariate data, was used for data analysis and visualization.47 (C) Heatmap of signaling pathway scores in KG-1 and Kasumi-1 cells (unsupervised hierarchical clustering; Euclidean distance; complete linkage). Signature scores were calculated as detailed in "Materials and methods." (D) Kaplan-Meier estimate of survival from diagnosis in TCGA-AML cases with abnormalities in DE genes between KG-1 (n 5 34) and Kasumi-1 cells (n 5 10). KM curves (median split of signature scores) were generated using GEPIA2, an enhanced Web server for TCGA gene expression profiling and interactive analysis (http://gepia2.cancer-pku.cn/#index). Signature scores are calculated as the mean value of log2 transcripts per million. GEPIA2 uses the log-rank (Mantel- Cox) test to compare survival distributions. pathways related to inflammatory responses, cellular response to immune infiltration, and expression of immune checkpoints and cytokine stimuli, response to stress, cytokine-cytokine receptor IFN signaling molecules (Figure 4A-B). Importantly, RFS and interactions, and interleukin-17 (IL-17)–mediated and tumor OS estimates were significantly worse for TCGA-AML patients necrosis factor–mediated signaling (Figure 3C; supplemental with abnormalities in query genes (median RFS and median OS, Table 6). 11.4 months) compared with patients without abnormalities in query genes (median RFS, 24.1 months; median OS, 27.0 The DE genes that we identified in primary BM samples were months; P 5 .0068 and P 5 .0009, respectively; Figure 4C). By further assessed in silico for potential prognostic value in TCGA- contrast, the 16 immune genes that were downregulated in AML cases. Data were accessed, analyzed and visualized using patients with TP53-mutated AML (supplemental Table 5) carried the cBio cancer genomics portal.34 Abnormalities of the 18 no prognostic significance (supplemental Figure 5). Taken immune genes that were overexpressed in patients with TP53- together, these findings suggest that the immunological TME mutated AML (by default, mRNA upregulation, amplification, of TP53-mutated AML is inherently proinflammatory and IFN-g deep deletion and missense mutations) significantly correlated dominant and that these molecular features are associated with with TP53 mutational status (P 5 .041; Figure 3D), higher poor clinical outcomes.

5018 VADAKEKOLATHU et al 27 OCTOBER 2020 x VOLUME 4, NUMBER 20 Table 2. Patient characteristics (immunotherapy cohort) with greater and lower than median gene expression. As shown in 5 All patients Patients with TP53 mutations Figure 5D, genes/proteins overexpressed in KG-1 AML (n 34; (N 5 45) and/or 17p abnormalities (n 5 15*) supplemental Table 7) predicted for shorter OS (HR, 1.7; log-rank 5 Age, median (range), y 61 (27-81) 61 (27-81) P .048). In contrast, genes upregulated in Kasumi-1 AML were unable to stratify patient survival (Figure 5D). Males/females, n 24/21 8/7 AML risk stratification We have recently shown that patients with IFN-g–dominant (2017 ELN), n (%) AML are less likely to respond to induction chemotherapy.15 It Favorable 3 (6.7) 0 (0) has also been reported that IFN-inducible genes mediate 35 Intermediate 8 (17.8) 0 (0) resistance to genotoxic damage in solid tumor cell lines. To Adverse 34 (75.6) 15 (100) mimic a proinflammatory TME, KG-1 and Kasumi-1 cells were treated with 100 ng/mL IFN-g for up to 24 hours. We initially Secondary AML, n (%) 15 (33.3) 7 (46.7) confirmed the expression of CD119, the ligand-binding portion of Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/20/5011/1779787/advancesadv2020002512.pdf by guest on 27 October 2020 No. of prior lines of therapy, 2 (1-9) 2 (1-4) median (range) the IFN-g receptor, on AML cell lines using flow cytometry (supplemental Figure 7A). Transcriptional changes in response to *BM samples from 13 out of 15 patients were available for immune gene expression exogenous IFN-g were prompter and more robust in Kasumi-1 cells profiling. All 15 patients with TP53 mutations/17p abnormalities were included in clinical analyses. compared with KG-1 cells, as indicated by the higher number of DE transcripts (supplemental Figure 7B). Both the expression of IFN- g target genes, including STAT1, NLRC5, IRF1,andISG15 (supplemental Figure 7C), and IFN-g–induced cytotoxicity (sup- Truncating TP53 mutations correlate with enhanced plemental Figure 7D) were higher in Kasumi-1 cells, suggesting IFN-g and inflammatory signaling in AML cell lines that cellular responses to IFN-g are blunted in KG-1 cells with heightened basal IFN-g signaling. Pretreatment with Prima-1MET, It has recently been shown that TP53 missense mutations in AML which restores the transcriptional transactivation function of mutant carry a gene signature of TP53 inactivation.32 Having previously TP53, was associated with induction of cell death in Kasumi-1 cells identified IFN-g–related microenvironmental signatures that corre- (median 62% reduction of cell viability compared with 21% in KG-1 late with chemotherapy resistance in AML,15 we performed in vitro cells), suggesting the specificity of Prima-1MET for TP53 reactivation modeling experiments with commercial AML cell lines to elucidate (supplemental Figure 7D). The occurrence of cytotoxicity after the potential impact of missense TP53 mutations on IFN signaling. TP53 reactivation with Prima-1MET was antagonized by exoge- DNA single-nucleotide variant amplicons, RNA and protein lysates nous IFN-g in Kasumi-1 cells, but not in KG-1 cells (supplemental were prepared as detailed in Materials and Methods. The inter-assay Figure 7D), indicating that Prima-1MET-treated Kasumi-1 AML reproducibility of RNA and protein measurements is shown in cells may receive prosurvival rather than cytotoxic signals as a result supplemental Figure 6A. KG-1 cells harbor a sequence change of IFN pathway activation. Finally, Kasumi-1 cells exhibited changes (c.67211G.A)thataffectsadonorsplicesiteinintron6oftheTP53 in gene expression in response to Prima-1MET, but not in response gene, resulting in a loss of protein function. Kasumi-1 AML cells harbor to the MDM2 inhibitor nutlin-3, including the upregulation of a known missense mutation of TP53 (R248Q). In agreement with this inhibitors of IFN signaling, such as SOCS1 and SOCS3,and knowledge, TP53 protein and RNA were not detected in KG-1 cells targets for transactivation by TP53,suchasFOS and PTGS2 (supplemental Figure 6B). The single-nucleotide variant assay (supplemental Figure 7E-F; supplemental Table 8). By contrast, confirmed the presence of TP53 (R248Q) mutations in control HuT- transcriptomic changes were minimal in Prima-1MET–treated KG-1 78 cells (data not shown), in accordance with available knowledge AML cells (supplemental Table 9). In aggregate, in vitro experi- from the COSMIC database (https://cancer.sanger.ac.uk/cosmic). ments suggest that truncating TP53 mutations may correlate with – A list of DE genes between KG-1 and Kasumi-1 cells was heightened IFN signaling and with resistance to IFN-g induced generated by considering false discovery rate ,0.01 and $1.7-fold cell cytotoxicity. change (Figure 5A). Specifically, KG-1 AML cells with a truncating, TP53-mutated patients with relapsed/refractory AML loss-of-function TP53 mutation overexpressed genes involved in show evidence of antileukemic activity from salvage IFN-g–mediated signaling and inflammation, including HGF, CIITA, immunotherapy with flotetuzumab PIM1, OSM, STAT1, and IRF1 (Figure 5B; supplemental Table 7). KG-1 cells also showed higher expression of PD-L1 and major We have previously shown that baseline IFN-g–related mRNA histocompatibility complex class I, which are known to be regulated profiles, including the TIS score, are associated with response to by IFN-g, at the protein level compared with Kasumi-1 cells flotetuzumab immunotherapy in patients with PIF and early- (supplemental Figure 6C). PI3K-Akt, NF-kB, JAK/STAT and TP53 relapse AML.15 We therefore correlated TP53 mutational status pathway genes, as well as genes associated with T helper 17 with immune landscapes and with complete responses to (Th17) differentiation, were significantly enriched in KG-1 cells flotetuzumab in a cohort of 45 patients with R/R AML, including (Figure 5C; supplemental Table 8). Interestingly, supplemental 15 patients with TP53 mutations and/or chromosome 17p Figure 6D highlights that 3 functions (pathways in cancer, PI3K-Akt deletions usually associated with loss of one allele of TP53 and signaling, and Th17 cell differentiation) were shared between DE mutation/loss of the other (Table 2). The immune landscape of genes in AML cells lines and DE genes in patients with TP53- 30/45 patients in this cohort has been presented in a previous mutated and TP53-WT AML. To assess whether the experimentally publication.15 Baseline BM samples for immune gene expres- derived gene/protein signatures could be of potential significance sion profiling were available in 13 patients with TP53 mutations for survival prediction, we split TCGA-AML cases into 2 subgroups and/or genomic loss of TP53; among these, 77% (10/13) had

27 OCTOBER 2020 x VOLUME 4, NUMBER 20 TP53 MUTATIONS AND IMMUNOTHERAPY RESPONSE IN AML 5019 A

TP53 status TP53 status 3 Response TP53 mut./17p abn. Glycolytic.activity 2 WT MHC2 CD45 1 IFN.downstream Response APM Immunoproteasome 0 Complete response Exhausted CD8 T cells No response Cytotoxic cells –1 Cytotoxicity Partial response CD8 T cells –2 Lymphoid cells T cells –3 Myeloid.inflammation Apoptosis TGF.beta Mast cells Complete response+ PDL2 B–cells Partial response Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/20/5011/1779787/advancesadv2020002512.pdf by guest on 27 October 2020 Treg cells No response Th1 cells C 25 2 NK cells P ( ) = 0.08 TIGIT Hypoxia 66.7% PD1 20 CTLA4 CD56dim NK cells NOS2 IDO1 15 DCs MAGEs 33.3% Stroma 60.0% Endothelial cells 10 PDL1 40.0% IL10 Number of patients Neutrophils 5 Myeloid cells Macrophages TIS Inflammatory.chemokines 0 IFN.gamma B7–H3 TP53-mut./17p abn. TP53-WT MMR.loss APM.loss Proliferation ARG1 JAK/STAT.loss Immune infiltration High Intermediate Low (50% TP53/17p) (31.8% TP53/17p) (20% TP53/17p) D B 300 TP53 mut./17p abn. CR 200 TP53-WT SD * 100 CR CR * 75 SD MLFS 50 PD 25 CR PR 0 CRi Time on treatment CRi -25 PD Time to HSCT Best BM (%) change -50 PR Time to death/last FU SD * Alive at last FU -75 PD

-100 0 3 6 9 12 15 18 21 24 Months from study entry E 8 P = 0.0016 8 P = 0.0186 8 P = 0.0016 10 P = 0.010

7 6 6 8

6 6 4 4 TIS score Treg score Treg CD8 score 4 5 2 2 PR PR PR PR CR,CRh,CRi,MLFS CR,CRh,CRi,MLFS CR,CRh,CRi,MLFS 2 CR,CRh,CRi,MLFS 4 score Inflamm. chemokine 0 0 Response NR Response NR Response NR Response NR 6 9 7 10 P = 0.0031 P = 0.045 P = NS P = NS

7 5 4 8 score

5 3 PD-1 score IFN- 2 PD-L1 score 6

3 1 Exhaustion score PR PR PR PR CR,CRh,CRi,MLFS CR,CRh,CRi,MLFS CR,CRh,CRi,MLFS CR,CRh,CRi,MLFS 0 1 -1 4 Response NR Response NR Response NR Response NR

Figure 6. Immune landscape and immunotherapy response in patients with relapsed/refractory AML with and without TP53 mutations and/or 17p abnormalities. (A) Heat-map of immune cell type-specific scores and biological activity scores in patients with relapsed/refractory AML treated with flotetuzumab immunotherapy. ClustVis, an online tool for clustering of multivariate data, was used for data analysis and visualization.47 (B) Waterfall plot depicting changes in BM blasts after cycle 1 of flotetuzumab in patients with TP53 mutations and/or 17p deletion (n 5 14; a BM sample was not available in 1 patient who progressed on treatment. (C) Response to

5020 VADAKEKOLATHU et al 27 OCTOBER 2020 x VOLUME 4, NUMBER 20 increased immune infiltration in the TME, and only 23% (3/13) was higher in patients with TP53 abnormalities from the flotetuzu- clustered in the immune-depleted subgroup (Figure 6A). Over- mab cohort. all response rate (ORR: CR, CRi, MLFS, or partial response) in patients with TP53 mutations and/or 17p abnormalities was Discussion 60% (9/15), with 47% (7/15) achieving complete response Our multicohort AML study shows that patients with mutations in , ( 5% BM blasts on study; Figure 6B). Individuals with TP53- TP53, the most commonly inactivated gene in human cancer, 1 mutated AML derived benefit from flotetuzumab immunotherapy exhibit higher CD8 T-cell infiltration and IFN-g signaling than AML when compared with patients with TP53-WT AML (60.0% ORR subgroups with other risk-defining molecular lesions, including 5 vs 33.3% ORR, respectively; P .08; Figure 6C), although this CHIP-related mutations in ASXL1, TET2,andDNMNT3A. difference failed to reach statistical significance, likely as Comparison of immune gene expression profiles between a result of low patient numbers. In the TP53-mutated patient primary BM samples from patients with TP53-mutated and subgroup, the reduction of BM blasts relative to baseline TP53-WT AML led to the identification of an immune gene set Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/20/5011/1779787/advancesadv2020002512.pdf by guest on 27 October 2020 averaged 51.2% (Figure 6B). Time on treatment and time to that is enriched in ontologies related to IFN-g/inflammatory patient death and/or censoring are summarized in Figure 6D for responses and IL-17/tumor necrosis factor–mediated signaling individuals with TP53 mutations and/or 17p deletion, including and that stratifies RFS and OS in a large cohort of TCGA-AML 3 patients who proceeded to receive allogeneic HSCT. In cases. Similar to previous studies that computed an expression patients harboring TP53 abnormalities who achieved a CR signature of TP53-mutated breast cancer,36 our TP53-related (CRi), median OS was 10.3 months (range, 3.3-21.3), suggesting immune gene set showed no overlap with known TP53 pathway that flotetuzumab immunotherapy may alleviate the negative genes, probably owing to variations in the type of mutants prognostic impact of TP53 mutations. Notably, 2 patients with analyzed. IL-17 can exert protumoral roles early in inflamma- TP53 abnormalities had exceptional responses to flotetuzumab, tion and cancer initiation.37 IL-17 could contribute to multiple . with a CR duration 6 months (Figure 6D). myeloma pathobiology by promoting tumor cell growth and colony formation, as well as increased myeloma growth in murine The TIS, inflammatory chemokine, Treg, CD8,IFN-g,andPD1 xenograft models.38 The correlation between TP53 deregulation gene expression scores, but not PD-L1 and markers of T-cell and IL-17 overexpression remains to be confirmed in larger AML exhaustion (CD244, EOMES, LAG3,andPTGER4), were series. significantly higher at baseline in responders compared with nonresponders (Figure 6E), highlighting the association be- In vitro modeling experiments using commercial AML cell lines tween response to T-cell engagers and a T-cell–inflamed TME. with known mutations of TP53 allowed us to identify a set of DE Interestingly, CD8B was the top-ranking gene associated with RNAs/proteins between KG-1 and Kasumi-1 cells and suggest response to flotetuzumab, with an area under the receiver that TP53 inactivation, which is frequent among TP53 missense operating characteristic curve value of 0.879 (supplemental mutants,32,39 is associated with the upregulation of IFN-g Figure 8A-B). The analysis of DE genes between responders pathway molecules, Th17 genes, and intermediates involved in with (n 5 8) and without (n 5 10) TP53 mutations and/or 17p JAK/STAT and PI3K-Akt signaling and the proinflammatory NF- deletion identified gene sets that were specifically upregulated kB pathway. Abnormalities of the DE RNAs/proteins in the KG-1 (IFNG, CD8B, NKG7,andSOCS1) or downregulated (CTNNB1 AML signature correlated with higher BM immune infiltration and and ANGPT1) in responders with TP53 abnormalities (supplemental TP53 mutations in TCGA cases and were prognostic, as Figure 8C-D). suggested by the significant separation of the survival curves. In sharp contrast, genes/proteins overexpressed in Kasumi-1 Finally, flow cytometry analyses using paired BM aspirates and cells harboring a missense mutation of TP53 were unable to blood samples from a subgroup of patients with (n 5 9) and without stratify survival in TCGA cases. Furthermore, IFN-g induced TP53 abnormalities (n 5 26) showed a trend toward higher 1 1 more rapid and robust transcriptional changes in Kasumi-1 cells percentages of BM CD8 T cells, but not blood CD8 T cells, in compared with KG-1 cells, with the latter also being less sensitive to individuals with TP53 mutations and/or 17p deletion (median IFN-g–induced cytotoxicity. These mechanistic findings are in line 45.2% vs 28.9%; P 5 not significant; supplemental Figure 9). The with previous reports that correlate an increased expression of expression of activation markers CD25 and HLA-DR was lower in g– 1 1 IFN- related mRNA signature scores with poorer OS esti- BM-resident CD8 and CD4 T cells from patients with TP53 mates in patients with AML treated with curative intent on a “7 abnormalities compared with TP53-WT AML (supplemental Fig- 1 3” chemotherapy backbone.15 Prolonged IFN-g signaling ure 10). Furthermore, the expression of IL-7 receptor (CD127), can also activate a multigenic program conferring resistance to a marker for long-living memory T cells, was lower in TP53-mutated DNA damaging agents, including radiotherapy and chemother- AML. In line with our findings in newly diagnosed TP53-mutated 40 1 1 apy, in preclinical models of solid tumors. Intriguingly, loss- AML, the frequency of GZMB-expressing CD8 and CD4 T cells of-function mutations of BRCA1, a tumor suppressor gene

Figure 6. (continued) flotetuzumab in relation to TP53 abnormalities (x2 test). (D) Swimmer plot showing time on treatment and time to death and/or censoring in relation to clinical response in the 15 patients with TP53 mutations and/or 17p deletion with genomic loss of TP53. Response criteria are described in "Materials and methods." (E) TIS, inflammatory chemokine, Treg cell, CD8, IFN-g, PD1, PD-L1, and exhaustion mRNA scores in baseline BM samples from patients with TP53 mutations and/or 17p deletion. Complete responses were defined as either CR, CR with partial hematological recovery, CRi, or MLFS. Data were compared using the Mann-Whitney U test for unpaired determinations. CR, complete remission; CRh, complete remission with partial hematopoietic recovery; FU, follow-up; OB, other benefit (.30% decrease of BM blasts relative to baseline); NR, no response; PD, progressive disease; SD, stable disease.

27 OCTOBER 2020 x VOLUME 4, NUMBER 20 TP53 MUTATIONS AND IMMUNOTHERAPY RESPONSE IN AML 5021 involved in DNA repair and maintenance of genome integrity, escape.46 Hence, patients with TP53 mutations might require have also been shown to correlate with hyperactive IFN-g combinatorial immunotherapy approaches that also target Treg signaling at baseline and with altered cellular responses of cells and immune checkpoints, either concomitantly or sequentially. breast and ovarian cancer cells to IFN-g, including partial resistance to cytotoxicity.41 In conclusion, our study shows that TP53 mutations in AML are associated with higher T-cell infiltration, expression of immune MET Treatment of Kasumi-1 AML cells with Prima-1 was associated checkpoints, and IFN-g–driven transcriptional programs. These with the upregulation of inhibitors of IFN signaling, including gene expression profiles that have previously been shown to enrich SOCS1 and SOCS3, suggesting that TP53 missense mutations, in patients with chemotherapy resistance correlate with disease 32 whichcarryagenesignatureofTP53 inactivation, may lead to control in response to flotetuzumab immunotherapy.15 Our findings enhanced IFN signaling. Pharmacological TP53 reactivation is therefore encourage further studies of T-cell–targeting immuno- actively being pursued in patients with AML. MDM2 is an E3 therapeutic approaches in larger series of patients with AML who ubiquitin ligase that binds to TP53 and induces its proteasomal harbor this molecular lesion. Downloaded from http://ashpublications.org/bloodadvances/article-pdf/4/20/5011/1779787/advancesadv2020002512.pdf by guest on 27 October 2020 degradation. Treatment with DS-5272, an inhibitor of the TP53- MDM2 interaction, in mouse models of AML was associated with Acknowledgments the upregulation of inflammatory and IFN-associated genes, including The authors thank Barbara Shepherd for providing medical writing PD-L1, and translated into enhanced antileukemia control.42 support. The Study Alliance of Leukemia (www.sal-aml.org) is Whether TP53 reactivation in patients with cancer is associated gratefully acknowledged for providing primary patient material and with immune-mediated therapeutic effects remains to be established clinical data. in future clinical trials. This work was supported by grants from the Qatar National Post-hoc analyses of our cohort of patients with R/R AML treated Research Fund (NPRP8-2297-3-494) and the John and Lucille van with flotetuzumab suggest that immunotherapy may be efficacious Geest Foundation (S. Rutella) and by the National Institutes of in the salvage setting in individuals with altered TP53 status, who Health, National Cancer Institute (Outstanding Investigator Award had a mean 51.2% reduction of BM blasts and showed complete R35 CA210084 [J.F.D.] and Research Specialist Award R50 responses in 47% of cases. CA211466 [M.P.R.]). Responders showed intermediate-to-high immune infiltration at baseline and higher TIS, inflammatory chemokine, Treg, IFN-g, and Authorship CD8 T-cell scores, but not PD-L1 and markers of T-cell exhaustion, Contribution: J.K.D.-M. and S. Rutella contributed to the study con- compared with nonresponders, suggesting that the presence cept and design; J.V., S. Reeder, S.E.C., T.H., and S. Rutella de- of TP53 abnormalities does not hamper the response to T-cell veloped methodology; I.A., A.S.A., J.G., M.J.W., M.A., J.M., F.R., K.S., engagers providing an inflammatory TME is present. Further- H.A., F.S., J.M.M., M.C., A.C., P.J.M.V., B.L., M.B., and J.F.D. acquired, more, both CD8B and IFNG were higher at baseline in responders consented, and managed patients and processed patient samples: with TP53 abnormalities compared with responders having C.L., J.V., S. Reeder, S.E.C., T.H., A.L., T.O.Y., M.P.R., G.A.F., F.S., TP53-WT AML. Genes previously implicated in T-cell exclusion J.M.M., P.J.M.V., B.L., I.G., M.B., J.F.D., J.K.D.-M., and S. Rutella an- from melanoma lesions, such as CTNNB1,43 and angiogenesis- alyzed and interpreted data; J.F.D. was principal investigator at mediated suppression of cell cytotoxicity (ANGPT1)were Washington University (St. Louis, MO); B.L. was principal in- preferentially expressed in TP53-WT AML, potentially account- vestigator at Erasmus University Medical Centre (Rotterdam, The ing for the observed differences in immune infiltration. Netherlands); C.L. and S. Rutella wrote the manuscript; C.L., J.V., R/R patients with TP53 abnormalities that responded to flotetuzu- S. Reeder, S.E.C., T.H., A.L., M.P.R., I.A., J.G., M.J.W., M.A., J.M., F.R., mab experienced a median OS of 10.3 months. Interestingly, 2 K.S., H.A., G.A.F., F.S., J.M.M., M.C., A.C., P.J.M.V., B.L., I.G., M.B., patients in the TP53-mutated cohort had exceptional responses to J.F.D., J.K.D.-M., and S. Rutella reviewed and/or revised the manu- flotetuzumab, with a CR duration .6 months. The molecular and script; and S. Rutella supervised the study. clinical determinants of extraordinary responses in patients with Conflict-of-interest disclosure: J.M. and J.K.D.-M. are employees 44,45 cancer are actively being investigated. Broader cohorts of of MacroGenics (Rockville, MD). S.E.C. is an employee of Nano- TP53-mutated AML will be needed to identify immune correlates of String Technologies (Seattle, WA). The remaining authors declare unusual outcome in response to flotetuzumab and other investiga- no competing financial interests. The patent “Bispecific CD123 3 tional immunotherapies. CD3 Diabodies for the Treatment of Hematologic Malignancies” Our results also point to the establishment of an inherently (provisional application; attorney docket number 1301.0161P3) immunosuppressive and IFN-g–driven TME in patients with TP53- was filed 25 July 2019 and assigned serial number 62/878 368. – mutated AML. Previously described gene expression based pre- ORCID profiles: A.L., 0000-0002-1978-6301; A.S.A., 0000- dictors of response to immune checkpoint blockade in solid tumors, 0003-0015-5902; T.O.Y., 0000-0002-3283-0370; B.L., 0000- 20,21 PD-L1 such as the TIS score, as well as immune checkpoints , 0001-8982-5217; S. Rutella, 0000-0003-1970-7375. TIGIT, and LAG3 and markers of immune senescence, were overexpressed in AML with TP53 mutations relative to other Correspondence: Sergio Rutella, John van Geest Cancer Re- molecular subtypes. Some of the characterized genes may search Centre, College of Science and Technology, Nottingham therefore contribute mechanistically to the poor prognosis associ- Trent University–Clifton Campus, Nottingham NG11 8NS, United ated with TP53-mutated AML through the induction of immune Kingdom; e-mail: [email protected].

5022 VADAKEKOLATHU et al 27 OCTOBER 2020 x VOLUME 4, NUMBER 20 References

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