5-Azacytidine Prevents Relapse and Produces Long-Term Complete Remissions in Leukemia Xenografts Treated with Moxetumomab Pasudo

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5-Azacytidine prevents relapse and produces long-term PNAS PLUS complete remissions in leukemia xenografts treated with Moxetumomab pasudotox

Fabian Müllera,b, Tyler Cunninghama,1, Stephanie Stookeya,2, Chin-Hsien Taia, Sandra Burkettc, Parthav Jailwalad,e, Maryalice Stetler Stevensonf, Margaret C. Came, Alan S. Wayneg, and Ira Pastana,3

aLaboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; bDepartment of Hematology/Oncology, University Hospital Erlangen, 91054 Erlangen, Germany; cMouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702; dAdvanced Biomedical Computing Center, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research Inc., Frederick, MD 21702; eCollaborative Bioinformatics Resource , Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; fLaboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and gChildren’s Center for Cancer and Blood Diseases, Division of Hematology, Oncology, and Blood and Marrow Transplantation, Children’s Hospital Los Angeles, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90027

Contributed by Ira Pastan, December 31, 2017 (sent for review August 16, 2017; reviewed by Francis Mussai and Daniel A. Vallera) Moxetumomab pasudotox (Moxe) is a chimeric protein composed of uniformly expressed (19), some HCL and two-thirds of ALL pa- an anti-CD22 Fv fused to a portion of Pseudomonas exotoxin A and tients do not respond to Moxe. To study resistance to Moxe, three kills CD22-expressing leukemia cells. It is very active in hairy-cell leukemia cell lines were treated in vitro until a resistant clone grew leukemia, but many children with relapsed/refractory acute lympho- out (15–17). We found that resistant CA46 cells had a small blastic leukemia (ALL) either respond transiently or are initially re- chromosomal deletion containing WDR85; the deletion was pre- sistant. Resistance to Moxe in cultured cells is due to low expression existing in the population of cells. Resistance in KOPN-8 and HAL- of diphthamide genes (DPH), but only two of six ALL blast samples 01 was due to promoter silencing of DPH1 and DPH4, respectively. from resistant patients had low DPH expression. To develop a more The resistance was associated with methylation of CpG islands in clinically relevant model of resistance, we treated NSG mice bearing the specific gene promoters, which was partially reversed by treating KOPN-8 or Reh cells with Moxe. More than 99.9% of the cancer cells the cells with the demethylating agent 5-azacytidine (5-AZA). were killed by Moxe, but relapse occurred from discrete bone mar- To identify mechanisms of resistance to Moxe in patients, we + row sites. The resistant cells would no longer grow in cell culture and studied primary ALL blasts from children with CD22 pre–B-ALL showed major chromosomal changes and changes in phenotype treated on the pediatric phase I trial (NCT01891981) and found with greatly decreased CD22. RNA deep sequencing of resistant resistance in most patients is not associated with low diph- KOPN-8 blasts revealed global changes in gene expression, indicat- thamide gene expression. To understand Moxe-resistance in ing dedifferentiation toward less-mature B cell precursors, and vivo, we then developed two ALL mouse models. In mice, we showed an up-regulation of myeloid genes. When Moxe was combined with 5-azacytidine, resistance was prevented and survival in- Significance creased to over 5 months in the KOPN-8 model and greatly improved in the Reh model. We conclude that Moxe resistance in mice is due to a new mechanism that could not be observed using cultured cells Moxetumomab pasudotox is a fusion protein of an anti-CD22 Pseudomonas and is prevented by treatment with 5-azacytidine. Fv and exotoxin. It is highly active against leukemia in vitro but acute lymphoblastic leukemia (ALL) patients often are resistant. Studies with cultured cells showed re- acute lymphoblastic leukemia | CD22 | immunotoxin | sistance is caused by reduced diphthamide, the intracellular Moxetumomab pasudotox | 5-azacytidine target of Pseudomonas exotoxin, but diphthamide is not reduced in most cells from most ALL patients. To study how re- reatment of childhood acute lymphoblastic leukemia (ALL) sistance develops in animals, we injected ALL cells into mice Tresults in 80% long-term remissions (1). Treating relapsed and found that resistant cells occur in discrete bone marrow ALL is a challenge and the likelihood of achieving a long-term niches and contain major chromosomal and transcriptional remission decreases dramatically with each relapse (2). Conse- changes. Mice pretreated with 5-azacytidine show greatly im- quently, novel therapies are needed to treat relapsed ALL more proved responses, supporting a trial of the combination in successfully (3). Emerging new treatment concepts include leukemia patients. engineered chimeric antigen receptor (CAR) T cells (4–6), – CD19-targeting BiTEs (7 9), B cell receptor (BCR)-signaling Author contributions: F.M. and I.P. designed research; F.M., T.C., S.S., C.-H.T., S.B., P.J., and M.S.S. inhibitors (10), or antibody drug conjugates (3, 11). performed research; M.S.S., M.C.C., and A.S.W. contributed new reagents/analytic tools; F.M., The recombinant immunotoxin Moxetumomab pasudotox (Moxe) P.J., M.S.S., M.C.C., A.S.W., and I.P. analyzed data; and F.M., A.S.W., and I.P. wrote the paper. consists of a CD22-targeting antibody fragment and the Pseu- Reviewers: F.M., University of Birmingham; and D.A.V., University of Minnesota domonas exotoxin A (12). After binding to CD22, Moxe is in- Cancer Center. ternalized, and is transported to the cytosol where it ADP Conflict of interest statement: A.S.W. and I.P. are coinventors on patents assigned to the NIH for the investigational products. A.S.W. has received research support, honorarium, ribosylates elongation factor 2 at a unique diphthamide residue and travel support from Medimmune; and honorarium and travel support from Pfizer, (13). The ADP ribosylation leads to protein synthesis arrest and Kite Pharma, and Spectrum Pharmaceuticals. MEDICAL SCIENCES cell death. The diphthamide moiety (DPH) is generated by seven Published under the PNAS license. enzymes (DPH1 through DPH7). The homozygous deletion of 1Present address: Medical Scientist Training Program (MSTP), Miller School of Medicine, the genes for DPH2,-4,or-5 (14) and DPH7/WDR85 (15) or a University of Miami, Miami, FL 33136. substantial decrease of DPH1 (16) or DPH4 (17) protein renders 2Present address: MD Program, UNC School of Medicine, University of North Carolina at cells resistant to immunotoxins in vitro. Chapel Hill, Chapel Hill, NC 27599. Moxe is highly active in 85% of patients with relapsed/re- 3To whom correspondence should be addressed. Email: [email protected]. ∼ fractory hairy-cell leukemia (HCL) (12) and in 30% of re- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lapsed/refractory childhood ALL (18). Even-though CD22 is 1073/pnas.1714512115/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1714512115 PNAS | Published online February 5, 2018 | E1867–E1875 Downloaded by guest on September 27, 2021 found resistant leukemia cells develop in discrete bone marrow died from ALL after 40 d (Fig. 2A). The regrowing KOPN-8 cells (BM) niches and contain major chromosomal and transcriptional are detected in the murine BM before they are detected in other changes. Because these cells do not grow in culture when re- organs (20). To assess response to Moxe, we monitored BM- moved from mice, this mechanism could not have been discov- disease burden after treatment (Fig. 2B). BM-infiltration on ered using cultured cells. day 8 was 4%. On day 16, 4 d after the last dose of Moxe, no leukemia was detected in six of eight mice; in the two remaining Results mice, 1 in 100,000 events was KOPN-8. However, 10 d later, all Reduced DPH-Enzyme Expression Correlates with Resistance in mice relapsed with an average BM-infiltration rate of 0.05%, Selected Patients. We previously found that CD22-expressing which rapidly rose to 43% on day 34. leukemia cell lines that were repeatedly treated with Moxe in To visualize where the relapsed cells are located, we generated vitro develop resistance caused by the loss or a substantial re- a KOPN-8 subclone (KOPN-8L) expressing GFP and luciferase duction of one of the enzymes required for the diphthamide (Fig. S1). This clone closely resembles the parental KOPN-8 in synthesis (15–17). To study whether primarily Moxe-resistant growth kinetics and sensitivity to Moxe. Eight days after in- ALL blasts from patients are resistant due to low DPH expres- travenous injection with KOPN-8L cells, mice showed bio- sion, we analyzed their expression by RNA deep sequencing in luminescence over the spine, hip, and calvarium (Fig. 2C). eight primary patient samples (Fig. 1). Compared with the av- Vehicle-treated mice showed a rapid increase in biolumines- erage expression in seven Moxe-sensitive cell lines, two of six cence correlating with disease progression. In Moxe-treated nonresponders showed a two- to eightfold reduced expression of mice, there was no bioluminescence detected 4 d after the DPH4 (Fig. 1A). However, the BM-blasts of Moxe-sensitive pa- last dose of Moxe. On day 29, relapse occurred as evidenced by tients also showed reduced DPH gene expression. To assess recurrence of the bioluminescence in both mice. The mice were whether the decrease is functionally relevant, we compared the retreated with a second cycle of Moxe. The bioluminescence average DPH-expression of the sensitive cell lines to the pre- fell, but persisted over the right tibia in mouse 2, indicating viously established resistant cell clones (Fig. 1B). A 17-fold re- local persistence of ALL cells. Mouse 2 was killed, the leg duction of DPH1 in KOPN-8-R (16) and a 10-fold reduction of paraffin-embedded, and stained with a human-specific mito- DPH4 in HAL-01-R (17) are sufficient to cause Moxe-resistance chondrial marker to identify human ALL. Dense ALL in- in vitro. The data indicate that for two patients, Moxe-resistance filtration was detected in the distal tibia (Fig. 2 D and E), but may have been due to reduced DPH4 expression. However, no ALL signal was present in the proximal femur or in the unlike as has been suggested by previous cell line studies, re- surrounding soft tissue (Fig. 2 D and F). Immunohistochemistry duced DPH enzymes were likely not involved in the Moxe re- confirmed local ALL persistence in the BM of the distal tibia. sistance of the other patients. Due to a lack of patient material, These results show that the majority of KOPN-8L cells die after these data were not validated by an independent method. initial Moxe treatment, but a few cells persist at an isolated BM site. KOPN-8 Relapse at Distinct BM-Sites Predates Systemic Relapse. To We then treated a group of KOPN-8L–bearing mice with two investigate resistance in an animal setting, we developed a NSG (Fig. 3A) or three (Fig. 3B) cycles of Moxe. The major signal on (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mouse model to study re- day 8 was found in close association with bone. Three doses of sistance in vivo, which we hypothesized would more closely re- Moxe abrogated the bioluminescence, but all Moxe-treated mice semble resistance in patients. NSG mice injected intravenously relapsed. The bioluminescence in relapse appeared over discrete with 5 million KOPN-8 cells die from systemic leukemia after a sites in bone, which were randomly distributed over the skeleton. median of 20 d. When treated with three doses of 0.4 mg/kg When the bioluminescence after the first cycle reached levels Moxe every other day from day 8, mice survived much longer but similar to day 8, mice received a second cycle of five doses Moxe

Fig. 1. Decreased DPH-gene expression correlates with resistance of one-third of the tested ALL samples. The reads for the indicated DPH enzymes were quantified by RNA deep sequencing of a total of seven Moxe-sensitive cell lines, normalized to reads per kilobase and million mapped reads, averaged, and set to 1. (A) The reads of the DPH-enzymes of six Moxe-resistant and two Moxe-sensitive patients were similarly determined by RNA deep sequencing and then normalized to the average of the seven sensitive cell lines. Nonresponder 6 was determined in duplicates all other patient reads were measured only once. (B) To determine the relative expression levels of the previously published resistant cell lines (16, 17), the DPH-expression was determined for their respective Moxe-sensitive cell line and multiplied by the changes in DPH for the resistant subclones, as determined by RT-PCR. The RT-PCR changes were determined in triplicates (16, 17).

E1868 | www.pnas.org/cgi/doi/10.1073/pnas.1714512115 Müller et al. Downloaded by guest on September 27, 2021 To test whether localized resistance occurred in a second an- PNAS PLUS imal model, we generated a GFP/luficerase-expressing RehL subclone (Fig. S2). The Reh cell line was chosen, because they are equally sensitive to Moxe as KOPN-8 (20), but distinct from the KMT2A-ENL translocated KOPN-8, the Reh cells harbor a TEL-AML1 translocation. Mice bearing RehL where treated with repeated cycles of Moxe (Fig. 3C). Vehicle-treated mice progressed as expected. The bioluminescence of RehL-bearing micetreated with Moxe fell after the first treatment cycle but the signal persisted at isolated spots over bones. If not treated again, the Reh-bearing mice developed hind-limb paralysis and splenomeg- aly and died of progressive systemic leukemia (Fig. S3). Mice receiving a second cycle of Moxe showed stabilization of the bioluminescence and bioluminescence increased 2 d after the third Moxe cycle. The RehL cells after the third Moxe cycle were considered Moxe-resistant. The two xenografts show that al- though Moxe is very active initially, a resistant subclone develops and persists at local BM sites, where it survives treatment until a systemically resistant clone emerges after several cycles of treatment.

Leukemia Cells That Acquired Resistance Showed Profound Changes in Phenotype. We attempted to grow the resistant KOPN-8L-R cells from mice in cell culture to facilitate their study. But the KOPN-8L-R cells would not grow, indicating that they had un- dergone major changes in the mice and suggesting that they had become dependent on the BM environment. Because in vitro generated Moxe-resistant cells show decreased expression of DPH enzymes, we assessed changes in mRNA levels by real-time PCR (Fig. 4A). The KOPN-8L-R had no decrease in any of the DPH-enzyme mRNAs; however, the CD22 mRNA was more than fourfold reduced. Flow cytometry confirmed the reduction of cell surface CD22 and of other B cell markers (Fig. 4B). The surface CD22 was reduced 2.5-fold (P < 0.0001), CD19 was reduced 2.1-fold (P = 0.0001), and CD10 was reduced 80-fold (P < 0.0001); surface CD28, however, increased 3.7-fold (P = 0.001). − No CD22 population was detected (Fig. S4). The total CD22 protein by Western blot in FACS-sorted cells from five resistant and sensitive mice was reduced sevenfold (Fig. 4C). Because surface CD22 was reduced only twofold, these data suggest a profound depletion of intracellular CD22 needed for replenishing cell surface CD22 (21). We also examined the resistant RehL-R cells that emerged after mice were treated with three cycles of Moxe and found they Fig. 2. Moxe treatment clears the BM from ALL but disease relapses from had acquired similar changes in surface markers as KOPN-8L-R discrete BM-sites. (A) Kaplan–Meyer survival curve for mice treated with three (Fig. 4D). We found the resistant cells had a 1.6-fold decrease of doses of 0.4 mg/kg Moxe every other day from day 8 or vehicle. The P value CD22, a 1.6-fold decrease in CD19, a 1.8-fold decrease in CD10, was determined by log-rank test. (B) Rate of BM infiltration for KOPN-8 bearing mice treated with the schedule in A and killed at indicated times. Symbols and a 3.6-fold increase in CD33. show individual mice, lines indicate means, error as SD, significance determined by unpaired t test, with P value as ns = not significant, ***P < 0.001. KOPN-8L-R Acquired Partial Moxe Resistance. Because the resistant (C) Mice were injected with KOPN-8L and treated with three doses of 0.4 mg/kg KOPN-8L-R cells alone cannot grow in culture, we hypothesized Moxe every other day or vehicle from day 8. Moxe-treatment was repeated that they need BM support to survive. We investigated whether from day 29 (red arrows). Bioluminescence signal was determined at treatment BM stromal cell lines would allow the resistant cells to grow and start (day 8) and at indicated days. All measurements were done at identical demonstrated that the murine calvarium-derived mesenchymal camera settings. Pictures shown are scaled from a radiance of 100–30,000. stromal cell line OP-9 rescued cell growth of KOPN-8L-R cells (D–F) Mouse 2 was killed, the right leg excised, formalin-fixed, paraffin- in vitro. Comparing KOPN-8L-R and KOPN-8L cells growing embedded, and stained for a human-specific mitochondrial marker (Mito-A/B, with OP-9 cell support, we found KOPN-8L-R had an IC of peroxidase signal). Images were digitalized using 40-fold optical magnification. 50 3.0 ng/mL, which was 10-fold higher than the IC50 of the sensitive KOPN-8L cells with or without OP-9 cells (Fig. 4E). These data every other day. The systemic bioluminescence fell below the show that KOPN-8L-R cells need stromal cell support to survive MEDICAL SCIENCES detection limit, but the signal over the local sites persisted, and and that the OP-9 support itself does not change the Moxe ac- then spread to produce a systemic bioluminescence in relapse. tivity against sensitive KOPN-8L cells. This suggests that the KOPN-8L-R cells acquired a partial and nonreversible resistance Mice not receiving a third cycle died from progressive disease. Of to Moxe. the three mice receiving a third cycle, two mice responded well but the systemic bioluminescence of the third mouse increased, Moxe-Resistant ALL Showed Major Chromosomal Changes. Because indicating a generalized Moxe-resistance after the second cycle. it is characteristic for ALL, we investigated whether the resistant These resistant cells were termed KOPN-8L-R. cells had developed additional chromosomal alterations and

Müller et al. PNAS | Published online February 5, 2018 | E1869 Downloaded by guest on September 27, 2021 Fig. 3. Leukemia relapse stays sensitive to retreat- ment for several cycles. Mice bearing KOPN-8L cells were treated with two (A), or three (B)cyclesofMoxe from days 8, 38, and 57, as indicated by the arrows. One cycle consisted of three doses of 0.4 mg/kg Moxe every other day. Bioluminescence was measured at indicated days after leukemia inoculation. All measurements were done at identical camera settings. Pictures shown are scaled from a radiance of 100–30,000. Shown are representative images of four independent experiments with similar results. (C) RehL-bearing mice were treated with three cycles of Moxe from days 6, 27, and 41. Mice were imaged at indicated days. All measurements were done at identical camera settings. Pictures shown are scaled from a radiance of 200–30,000 with the exception of days 48 and 59, which were scaled from a radiance of 1,000–30,000 to reduce background.

found many changes (Fig. 4F). The parental KOPN-8L cells In addition, there was an increase in the monocyte-specific genes showed the expected karyotype of 44,XX, t(11;19), t(13;14), −13, TLR7 (5.2-fold), TNFRSF21 (=DR6, 14.4-fold), PLEK (8.8-fold), and −14. The KOPN-8L-R cells showed additional changes in- CSF2RB (=GM-CSF-R, 1.9-fold), and FLT3 (1.6-fold), and the cluding trisomy of chromosomes 6, 7, 8, and 20 and two addi- activation marker CD28 (>50-fold). tional t(5;8)-chromosomes, resulting in a 50,XX karyotype. Because smaller but concordant changes in several genes of These chromosomal changes could be responsible for the one pathway can be biologically relevant, the changes were an- changes in phenotype of KOPN-8L-R. In addition, we found that alyzed using gene-set enrichment analyses (GSEA) (23). The the Moxe-resistant RehL-R cells from three individual mice also changes were ranked from most up-regulated to most down- showed major changes in their karyotype by G-band staining (Fig. regulated and compared with the “c2-curated pathway” gene S5). Compared with the Moxe-sensitive RehL, the resistant sub- sets and the “c7-immunological” gene sets provided by the clone of one mouse had loss of chromosomes 2 and 18, while the Molecular Signatures Database (MSigDb). GSEA generates the subclone of another had gained chromosomes 2 and 16, and a q-value describing the chance that differences between genes in subclone of a third mouse had gain of chromosomes 2 and 3 and the experimental list and a curated list are false. A low q-value loss of chromosomes 18, 19, 21, and 22. The predominant resistant indicates a high probability that the GSEA result is not found by subclone of each mouse presented distinct karyotype changes, chance. In addition, GSEA provides the enrichment score, which which were different from the untreated cells growing in mice. is generated by an algorithm that increases when a gene is pre- Because the KMT2A-rearrangement distinguishes the KOPN- sent in both the experimentally determined list and the MSigDb 8 cell line from other ALL subtypes (22), we determined if ex- list, or decreases when it is not. The enrichment score (ES) is pression of the KMT2A-fusion gene changed significantly in then normalized (NES) (23). resistant KOPN-8 cells. Analyzing the fusion transcript expres- Fig. 5B shows four gene sets that closely resemble the differ- sion from the RNA deep-sequencing data, we found no differences between KOPN-8L-R and KOPN-8L. Genes that are ence between sensitive and resistant cells (Fig. 4G). On average, down-regulated in large prelymphocytes compared with mature there were 13 uniquely aligned reads for KOPN-8L cells and B-lymphocytes (24) are down-regulated in KOPN-8L-R (q < 12.8 reads for KOPN-8L-R cells (P = 0.74). The major changes 0.0009, NES = −2.33). Genes defined as “BCR pathway” (25) in karyotype did not alter the expression level of the KMT2A- (q = 0.004, NES = −2.07) are also down-regulated. Both sets of fusion mRNA. data suggest a change of KOPN-8L-R to less mature B-cells. Additionally, genes that are down-regulated in B-cells com- RNA Deep Sequencing Identifies Down-Regulation of B Cell-Specific pared with plasmacytoid dendritic cells (pDC) (26) were up- Gene Sets. To study effects of the chromosomal and other regulated in KOPN-8L-R (q < 0.0009, NES = 2.15), indicating changes in the resistant ALL cells, we analyzed our RNA deep- a change toward pDCs. In addition, genes up-regulated in B cells sequencing data on flow-sorted ALL cells from five mice bearing compared with monocytes (26) were down-regulated in KOPN- KOPN-8L and KOPN-8L-R. The dot-plot in Fig. 5A shows the 8L-R cells, indicating a change toward a monocytic expression overall distribution of the average log2 (counts per million) for the pattern (q = 0.003, NES = −2.04). Taken together, GSEA 16,561 genes expressed in KOPN-8L and KOPN-8L-R. In accord analysis supports a dedifferentiation of KOPN-8L-R cells from with the flow data, there is a substantial decrease in mRNA for pre-B cells toward less-mature B cells and cells of non-B MME (=CD10, 16-fold) and MS4A1 (=CD20, 28-fold) and a less hematological lineages. than threefold but significant (P < 0.001) decrease for CD19 (1.7- The ingenuity pathway analysis (IPA) further supported a fold), CD22 (2.2-fold), CD79A (2.2-fold), and CD79B (2.8-fold). change toward less-mature B cells, as indicated by the decreased

E1870 | www.pnas.org/cgi/doi/10.1073/pnas.1714512115 Müller et al. Downloaded by guest on September 27, 2021 PNAS PLUS

Fig. 4. Moxe-resistant relapse shows major phenotype and chromosome changes. KOPN-8L and KOPN-8L-R were engrafted in five mice each, their BM sorted for ALL cells, which were split to produce total RNA and total protein. (A) mRNA levels by real-time PCR in KOPN-8L-R from BM or control KOPN-8-R from cell culture (CC) (16) relative to KOPN-8L (dotted line). Bars indicate average fold-changes of CT-values in triplicates, error as SD. P values as ns = not significant, *P < 0.033, **P < 0.002, ***P < 0.001, ****P < 0.0001. (B) The BM-derived ALL blasts from five independent mice bearing KOPN-8L or KOPN-8L-R were measured for the indicated markers by flow cytometry, t tests determined P values as ns = not significant, *P < 0.033, **P < 0.002, ***P < 0.001. (C) Western blot for total CD22 protein of BM-derived ALL blasts from five independent mice, densitometry values are actin normalized. (D) BM from three individual mice bearing RehL or RehL-R (from Fig. 3C) were analyzed as described in B.*P < 0.033, **P < 0.002, ***P < 0.001. (E) KOPN-8 GL (GL) cells were cultured alone or GL cells and GL- + R cells were cultured with OP-9 stromal cells, treated with Moxe, viability determined by flow cytometry for the GFP cells, which was normalized to untreated control (100%) and 0% is a true 0. Each symbol represents the mean percentage of living cells in triplicates, error as SD. (F) Chromosome analysis by spectral karyotyping showing the karyotype of KOPN-8L [44,XX, add(8q), t(11;19), t(13;14), −13, −14] acquired six additional chromosomal aberrations in KOPN-8L-R [+6, +7, +8, +t(8;5) ×2, and +20]; >10 karyotypes were analyzed, shown is the representative. (G) The expression level of the fusion transcript of the KMT2A (chromosome 11) and the MLLT1 (chromosome 19) translocation was measured by RNA deep sequencing, as described in Materials and Methods. Each symbol

represents the number of uniquely mapped reads of individual mice, bars represent the average, error as SD, P values were determined by unpaired t test. MEDICAL SCIENCES

expression of BCR-pathway genes. IPA identified the BCR- specific TFs TCF3 (−1.5×, P < 0.0001), FOXO1 (−3.2×, P < pathway as the second-most altered canonical pathway in 0.0001), EBF1 (−1.5×, P < 0.0001), and BCL6 (−6.3×, P < 0.0001) KOPN-8L-R compared with KOPN-8L (Fig. S6). decreased, while DC-determining TFs ID2 (2.4×, P < 0.0001), The transcription factors (TFs) determining early and in- IRF4 (2.1×, P < 0.0001), and GFI1 (>50-fold, P < 0.0001) in- termediate B cell fates also changed significantly in agreement creased (Fig. 5 A and C) (28). Together, these data indicate that a with the pathway changes (Fig. 5C) (27). We found the B cell- significant down-regulation of several key TFs in B cell development

Müller et al. PNAS | Published online February 5, 2018 | E1871 Downloaded by guest on September 27, 2021 0.3 mg/kg Moxe intravenously from day 8. Untreated mice progressed rapidly (Fig. 6 A and B, Upper). The bioluminescence for mice treated with Moxe alone fell and rose again in relapse as expected. Daily treatment with 5-AZA alone slowed disease progression, as indicated by the lower bioluminescence on day 8 (P = 0.003). The bioluminescence then increased between days 8 and 19 (P = 0.06). The combination of Moxe and 5-AZA resulted in a total abrogation of the bioluminescence and none of the mice showed any luciferase signal thereafter. The changes in bioluminescence correlated with animal survival (Fig. 6B, Lower). Animal survival was marginally prolonged by 5-AZA alone, and significantly increased by Moxe alone. However, mice that received the combination treatment were healthy and disease-free on day 120, when the experiment was terminated. Pathological examination and BM flow at day 120 showed no evidence of leukemia. It appeared that these mice were cured.

5-AZA Did Not Reverse Established Moxe-Resistance. To determine whether 5-AZA could overcome the established resistance of KOPN-8L-R in mice, we harvested ALL cells from mouse 5, reinjected the cells in new mice, and tested the effect of Moxe, 5- AZA, or the combination (Fig. 6C, Upper). Untreated mice showed a rapid increase in bioluminescence and died on day 25. Mice treated with 5-AZA from day 3 showed a slow, but steady increase in bioluminescence (P < 0.001). The combination of 5- AZA and Moxe slowed the increase of bioluminescence, but all mice progressed after the treatment ended. The mice treated with 5-AZA survived on average 4 d longer, Moxe-treated mice 11 d longer, and mice treated with the combination 16.5 d longer Fig. 5. RNA-deep sequencing confirms profound changes in KOPN-8L-R. than untreated mice (Fig. 6C, Lower). None of the mice showed BM-derived cells were FACS-sorted and their RNA isolated for RNA-deep long-term disease-free survival, as seen in the mice bearing sequencing. (A) The mapped reads were counted, normalized to counts KOPN-8L cells. These results indicate that once resistance is per million (cpm), log2-transformed, and each individual gene from resistant established it cannot be reversed with 5-AZA. cells (Res, x-coordinate) plotted against sensitive cells (Sens, y-coordinate). Every dot represents the average log (cpm) from five independent mice. 2 5-AZA Enhanced the Activity of Moxe in RehL Xenografts. Mice Highlighted are B cell marker genes (blue rhombi) that are down-regulated and non-B cell marker genes (red stars) that are up-regulated in KOPN-8L- bearing the RehL cells were treated as the mice bearing KOPN- Res. (B) All genes were preranked using limma’s t-statistic from most up- 8L. Vehicle-treated mice showed an increase in bioluminescence regulated to most down-regulated and the list was compared with (Fig. 7A). In mice treated with 5-AZA, bioluminescence in- MSigDb gene lists. The total number of genes and the number of over- creased between days 8 and 20 (P = 0.02). In mice treated with lapping genes between a pathway and the experimentally determined gene Moxe alone, bioluminescence on day 20 was stable compared list are indicated. Every gene present in the preranked gene list and in the with day 8 (P = 0.94). Only the Moxe/5-AZA combination greatly respective pathway is represented by a black line above the red (up- reduced the signal intensity on day 20 in all 10 mice (P = 0.001). ’ regulated) to blue bar (down-regulated), indicating the gene s rank in the The signal fell below the detection limit in four mice and the mice preranked list. The enrichment score (ES) (y axis) is generated by an algo- stayed free of bioluminescence up to 50 d. Nevertheless, all mice rithm, which increases a “running-sum statistic” when a gene is present in the preranked list or decreases when the gene is absent [green line (23)]. The relapsed from discrete sites closely associated with bones. Un- enrichment score is then normalized to the gene set size, resulting in the NES treated mice showed a median survival of 37 d, mice treated with (23). The NES and the false-discovery rate “q” are indicated. DN, down- Moxe of 45 d, 5-AZA treated mice of 51 d, and mice receiving the regulation; PID, pathway interaction database; UP, up-regulation. (C) combination of 67 d (Fig. 7B). Shown are TFs central to the early development of B cells, common lymphoid progenitor (CLP), lymphoid-primed multipotent progenitor (LMPP), multi- Discussion potent progenitor (MPP), and prepro-B cells. Indicated by dashed arrows are We have developed a mouse model of resistance of ALL cells to interactions between the TF driving development toward B cells or DCs. The Moxe, to understand the patho-biologic basis for reduced clinical fold up- (blue) and down-regulation (green) of TFs in KOPN-8L-Res are shown. Graph adapted from ref. 27. responsiveness. We find that the ALL cells growing in murine BM become resistant to Moxe and that the resistant cells have developed major chromosomal changes and changes in gene lead to a marked down-regulation of B cell-specific genes (27), expression, indicating dedifferentiation toward less mature B cell whereas the up-regulation of ID2, IRF4, and GLI1 likely result in precursors. These changes were not observed when Moxe- the up-regulation of genes expressed in monocytic cells. resistant cells were selected in cell culture, probably because the resistant cells from mice cannot grow in vitro. The Combination of 5-AZA and Moxe Prevented Local Resistance. An unexpected feature of the resistant cells growing in mice, Moxe-resistance in vitro can be caused by methylation of pro- which is more likely to recapitulate resistance in patients in comparison with in vitro cell line studies, is that resistant cells moter regions, which can be prevented by 5-AZA (16, 17). first appear at discrete locations in various bones and then Consequently, we hypothesized that the resistance observed in spread to other locations. Treatment initially clears the dissem- mice might also be due to DNA methylation and we tested 5- inated cells, which then regrow, originating from discrete BM- AZA in our model. Considering that the methylation changes sites and gradually become resistant. This behavior indicates that might occur early after cell implantation, we treated mice with resistance develops in several steps. It is also consistent with a daily doses of 5-AZA from day 3 (29) followed by five doses of protective BM-niche providing local supportive signals (10, 30,

E1872 | www.pnas.org/cgi/doi/10.1073/pnas.1714512115 Müller et al. Downloaded by guest on September 27, 2021 targeting BiTEs (37, 38) or CD19-targeting CAR-T cells (39, PNAS PLUS 40) similarly present with the loss of CD19, which is accompa- nied by a lineage switch to a myeloid leukemia not expressing B cell markers with additional chromosomal changes (37–40). For aggressive B cell malignancies, such chromosomal changes in relapse have been described as linear evolution out of the predominant clone at first diagnosis (41–43). Our model suggests that these chromosomal changes develop step-wise and by chance, and take place in cells that are protected by discrete BM- niches. Therapeutic pressure by repeated cycles of CD22- targeted immunotoxin gives rise to a linear subclone with substantial chromosomal and phenotypic changes. The therapy-resistant subclones of both, the KMT2A-ENL translocated ALL cell line KOPN-8 and the TEL-AML1 translocated cell line Reh, have a marked down-regulation of B cell- specific markers, including CD22 the target of Moxe. An only twofold reduction of the surface CD22 is paralleled by a profound reduction of intracellular CD22. The surface CD22 is internalized after receptor binding and then replenished by the intracellular pool (21). Thus, the reduction of the intracellular pool of CD22 may affect the internalization of Moxe over time. In accord with our previous data showing that CD22-targeted immunotoxins are more active the longer the cells are exposed (20, 44), the reduction of intracellular CD22 suggests that the resistant cells must be exposed to Moxe longer for them to die. Because Moxe has a short 20-min half-life in mice, the drug concentration after a bolus dose falls rapidly to low levels not capable of killing the resistant cells (20, 44). Even though suggested by our data, CD22 expression levels and Moxe activity does not necessarily correlate (45) and CD22-targeted immunotoxin can be highly active against ALL that expresses little CD22 (34). In summary, we showed in two mouse models that Moxe resistance is due to major structural changes in chromosomes and in gene expression. Systemic resistance develops after several cycles of treatment. By combining Moxe with 5-AZA, resistance and subsequent relapse is completely prevented in KOPN-8 xenografts and delayed in Reh xenografts. The marked increase of Moxe efficacy by 5-AZA makes this combination a promising approach for future clinical testing in children and adults with ALL. Fig. 6. The combination of 5-AZA and Moxe greatly improves responses of Materials and Methods the KOPN-8L xenografts. Mice inoculated with KOPN-8L were treated with vehicle, five doses of 0.3 mg/kg Moxe every other day from day 8, 2 mg/kg 5- Cell Lines. KOPN-8 and Reh cells were described previously (20). Cellular AZA from day 3, or the combination. Bioluminescence was determined be- identity was established by short tandem-repeat analysis. Cells were grown in RPMI-1640 with 10% FBS, 100 U penicillin, and 100 mg streptomycin. fore treatment start on day 8 and at indicated days. All measurements were + done at identical camera settings. Pictures shown are scaled from a radiance Generation of luciferase KOPN-8L and RehL cells is described in Figs. S1 and of 100–30,000. Mice shown in A and B were inoculated with KOPN-8L and S2, respectively. The Moxe-resistant derivate KOPN-8L-Res emerged spon- mice shown in C. with KOPN-8L-R. Symbols in B and C, Upper represent the taneously in vivo after repeated cycles of treatment, as described in Results. average bioluminescence intensities per mouse in the respective treatment The stromal cell line OP-9 (CRL-2749) was purchased from ATCC. group; P values were determined by t test. The respective Kaplan–Meyer For coculture experiments, OP-9 cells were seeded at 3,000 cells per well in survival graphs are shown in the in B, Lower, for KOPN-8L mice and C for a 96-well plate on day 0. On day 1, 50,000 GL-Res or GL cells were added and KOPN-8L-R; P values were determined by log-rank test. t test-derived P 6 h later treated with Moxe at indicated concentrations. The cells were values as ns = not significant, *P < 0.033, **P < 0.01, ****P < 0.0001. stained 48 h later with Annexin V/7-AAD and viability determined by flow cytometry.

31) and inducing a therapy-resistant state (32, 33). BM niche- Reagents. Moxe was supplied by Medimmune. 5-AZA (Sigma) was dissolved in induced Moxe resistance is in line with findings in pediatric ALL 0.9% NaCl at 2 mg/mL, sterile-filtered, and stored at −80 °C. Panobinostat patients treated with anti-CD22 immunotoxin, where clearance (Selleck) was dissolved in 2% DMSO, 2% Tween80, 48% PEG-300, and 48% water at 5 mg/mL. of peripheral blood blasts was observed despite persistence of Secondary Western blot antibodies were purchased from Santa Cruz,

BM disease (34). Because the development of resistance is polyclonal α-CD22 was produced in rabbits. Antibodies for flow cytometry MEDICAL SCIENCES prevented by treatment with 5-AZA, but 5-AZA is not effective (α-CD10-Cy5.5, α-CD19-PE, α-CD22-PE, α-CD28-APC, α-CD33-PerCP, and after resistance has developed, we believe that early changes in α-CD34-PE) were purchased from Becton Dickinson. gene-expression patterns contribute to the Moxe-resistance and possibly to the major chromosomal changes observed in cells Animal Studies. Animals were handled according to NIH guidelines; studies were approved by the National Cancer Institute Animal Care and isolated after several cycles of treatment. Use Committee. Relapse of ALL after chemotherapy commonly presents with Five million KOPN-8L (20), 1 million KOPN-8L-Res, or 5 million RehL cells chromosomal changes resulting in substantial phenotype changes (34) were injected on day 1 via tail vein into 6- to 8-wk-old NSG mice. Moxe (35, 36). Relapse of ALL after targeted therapy with CD19- was given intravenously, as indicated. 5-AZA was given at 2 mg/kg and

Müller et al. PNAS | Published online February 5, 2018 | E1873 Downloaded by guest on September 27, 2021 Fig. 7. 5-AZA enhances Moxe activity in the RehL xenograft. (A) Mice were inoculated with RehL cells expressing GFP/luciferase and treated with vehicle, five doses of 0.3 mg/kg Moxe every other day from day 8, 2 mg/kg 5-AZA intraperitoneally from day 3, or the combination. All measurements were done at identical camera settings. Pictures shown are scaled from a radiance of 100–30,000. (B) Kaplan–Meyer survival graph for mice in A; P values were determined by log-rank test as ns = not significant, ***P < 0.001, ****P < 0.0001.

panobinostat at 5 mg/kg intraperitoneally daily from day 3 until the last dose normalization and differential expression was carried out using limma-voom of Moxe (29). To assess BM response, mice were killed on days indicated. For (48). For GSEA, the genes were preranked by t-statistic and compared with survival studies mice were followed until they showed disease progression and curated gene lists in “pre-ranked mode” (23). To generate the reads corre- were killed. Disease progression for KOPN-8L-bearing mice was defined sponding to the KMT2A-ENL fusion, the BOWTIE aligner for gene mapping as >10% loss of highest body weight, for RehL-bearing mice as development (49) was combined with the BLAT and STAR aligner for mapping the genes of hind-limb paralysis or weight loss >10% of highest body weight. to the fusion junction (50). BM was extracted with mortar and pestle, mesh-filtered, washed, and either viably frozen or sorted with a FACS Aria (for FSC/SSC, FSC-H/FSC-W, and Patient Studies. Primary ALL blast samples from patients with CD22+ pre–B- pos GFP ). Six million sorted cells per mouse were split in half and lysed for ALL treated on a phase I trial of Moxe (NCT01891981) were collected with extraction of total RNA using the RNEasy kit (Qiagen) or lysed in modified informed consent under protocol 04-C-0102 and approved by the National · RIPA buffer [50 mM Tris HCl pH 7.5, 0.25% Na-Deoxycholate, 1% Nonidet P- Cancer Institute Institutional Review Board. Total RNA was extracted and 40, 150 mM NaCl, HALT protease inhibitor (Pierce)]. Total RNA was quality RNA deep sequencing performed as described above. controlled and samples with a registrant identification number of 10 sent to the National Cancer Institute RNA deep-sequencing facility; 1 μg was Luciferase Imaging. D-luciferin (75 mg/kg, VivoGlo; Promega) was injected reverse-transcribed and amplified with the QuantiFast SYBR Green PCR kit intraperitoneally, mice anesthetized with Isoflurane, and images were taken (Qiagen) for qRT-PCR using primers, as indicated in Table S1. The data were 5 min after injection using a Xenogen IVIS-100 (Caliper). analyzed using the comparative CT method (ddCT) with SDS manager (Ap- plied Biosystems) and normalized to the average CT values of three housekeeping genes. Statistics. Statistical analyses were performed with Graph Pad Prism v7.00 as unpaired t tests (two group comparison), as ANOVA (multiple comparison For immune-phenotyping, cells were Fc-receptor blocked, stained in PBS/ 1% BSA/2 mM EDTA/0.1% Na-Azide, analyzed on a FACS Calibur, and analyses), or as log-rank test (animal survival). quantified using FlowJo (TreeStar). For immunohistochemistry, murine tissue was formalin-fixed for 24 h, ACKNOWLEDGMENTS. The authors thank the Building 37 Animal Facility for stored in 70% ethanol for 48 h, and paraffin-embedded; 4-μm sections were their support. This work utilized the computational resources of the NIH stained with anti-human Mito A/B. High-Performance Computing Biowulf cluster (https://hpc.nih.gov/). This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. F.M. was supported in RNA Deep Sequencing. RNA libraries were prepared using the TruSeq stranded part by the German Research Foundation, Award MU 3619/1-1 and the In- RNA protocol and sequenced on an Illumina HiSeq2500 sequencer using terdisciplinary Center for Clinical Research, Erlangen, Award IZKF-J59 and TruSeq V4.0. Quality was checked using FastQC, reads were trimmed using IZKF-P002. A.S.W. was supported in part by National Cancer Institute Award Trimmomatic, and mapped to the human genome (hg19) by STAR in “2-pass” P30CA014089. The content is solely the responsibility of the authors and mode (46). The reads at gene level were quantified using subread feature does not necessarily represent the official views of the National Cancer In- counts (47) and normalized to library size as counts per million. Quantile stitute or the National Institutes of Health.

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