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(12) United States Patent (10) Patent No.: US 9,683,995 B2 Zon Et Al

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(12) United States Patent (10) Patent No.: US 9,683,995 B2 Zon Et Al US009683995B2 (12) United States Patent (10) Patent No.: US 9,683,995 B2 ZOn et al. (45) Date of Patent: Jun. 20, 2017 (54) METHOD OF TREATMENT OF SETDB1 2600/158 (2013.01); G0IN 2333/91011 EXPRESSING CANCER (2013.01); G0IN 2333/91017 (2013.01) (58) Field of Classification Search (71) Applicant: CHILDRENS MEDICAL CENTER None CORPORATION, Boston, MA (US) See application file for complete search history. (72) Inventors: Leonard I. Zon, Wellesley, MA (US); (56) References Cited Craig Ceol, Sherborn, MA (US); Yariv J. Houvras, New York, NY (US) FOREIGN PATENT DOCUMENTS (73) Assignee: THE CHILDRENS MEDICAL EP 2813571 12/2014 ........... C12N 15,113 CENTER CORPORATION, Boston, WO 2009,147445 12/2009 MA (US) OTHER PUBLICATIONS (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 Rodriguez-Paredes M. et al. Gene amplification of the histone U.S.C. 154(b) by 0 days. methyltransferase SETDB 1 contributes to human lung tumorigenesis. Oncogene 33, 2807-13, 2014.* (21) Appl. No.: 14/701,810 Li et al., J Biol Chem, 281 (28): 19489-19500 (2006). “The histone methyltransferase SETDB1 and the DNA methyltransferase (22) Filed: May 1, 2015 DNMT3A interact directly and localize to promoters silenced in cancer cell.' (65) Prior Publication Data Spannhoff. The International Journal of Biochemistry & Cell Biol ogy, 41 (Issue 1):4-11 (2009). “Cancer treatment of the future: US 2015/O293,099 A1 Oct. 15, 2015 inhibitors of histone methyltransferases.” Sun et al., Liver Int. 27(8): 1021-38 (2007). "Oncoproteomics of Related U.S. Application Data hepatocellular carcinoma: from cancer markers' discovery to func tional pathways.” (63) Continuation of application No. 13/147,713, filed as Wang et al., Molecular Cell, 12:475-487 (2003). “mAM facilitates application No. PCT/US2010/022994 on Feb. 3, conversion by ESET of dimethyl to trimethyl lysine 9 of histone H3 2010, now Pat. No. 9,040,286. to cause transcriptional repression.” Watanabe et al., Cancer Cell International, 8(15): 1-12 (2008). (60) Provisional application No. 61/260,476, filed on Nov. "Deregulation of histone lysine methyltransferases contributes to 12, 2009, provisional application No. 61/149,498, oncogenic transformation of human bronchoepithelial cells.” filed on Feb. 3, 2009. * cited by examiner (51) Int. C. GOIN 33/574 (2006.01) Primary Examiner — Elly-Gerald Stoica A 6LX 39/395 (2006.01) (74) Attorney, Agent, or Firm — Nixon Peabody LLP: C07K 6/40 (2006.01) David S. Resnick; Tari W. Mills CI2N IS/II (2006.01) CI2O I/68 (2006.01) (57) ABSTRACT CI2N IS/IT3 (2010.01) Provided herein are methods for diagnosing cancer by (52) U.S. C. determining the level of expression of SETDB1 in a bio CPC. G0IN 33/5743 (2013.01); A61K 39/39558 logical sample. Also provided herein are methods for treat (2013.01); C07K 16/40 (2013.01): CI2N ing cancer by administering an inhibitor of SETDB1 to a 15/1137 (2013.01); C12O 1/6886 (2013.01); subject in need thereof. CI2Y 201/0104.3 (2013.01); G0IN 33/574 (2013.01); C12N 23 10/14 (2013.01); C12O 12 Claims, 14 Drawing Sheets U.S. Patent Jun. 20, 2017 Sheet 1 of 14 US 9,683,995 B2 Figurel B. Figure C. 100 g - miniCoopR-SETDB1. 8O - miniCoopR-EGFP d 60 s E 40 f D S. 20 O 5 10 15 2O 25 Age (weeks) U.S. Patent Jun. 20, 2017 Sheet 3 of 14 US 9,683,995 B2 Figure 3. Figure58. intermediate High U.S. Patent Jun. 20, 2017 Sheet 5 of 14 US 9,683,995 B2 Figure 5. GFP Tumors SETDB1. Tumors SETDB1. is us Tubulin up wene up us um U.S. Patent Jun. 20, 2017 Sheet 6 of 14 US 9,683,995 B2 Figure 6. o N cy H d f N OO S- s > > s s s S 2 Anti-SETDB1. (AbCam 1:500) Anti-Tubulin (Cell Signaling 1:100) U.S. Patent Jun. 20, 2017 Sheet 7 of 14 US 9,683,995 B2 Figure 7. Ficure 3 f 5 gure 1A F. G F f : C F C G T R G C WM1968 BRAF wild-type 600 5 GCT ACA GTG AAA TCT, 3' CGA TGT CAC TTT AGA A. T W K S Figure 7S. WM262 BRAF V6OOE mutant 600 5 GCT ACA GAA AAA TCT 3' CGA TGT. CTT TTT AGA A. T E. K S Figure 7C A375 BRAF W600E mutant 6OO 5' GCT ACA GAG AAA, TCT 3' CGA TGT CTC TTT AGA A. T E K S U.S. Patent Jun. 20, 2017 Sheet 8 of 14 US 9,683,995 B2 Figure x. Figure 3A. 80000 160000 and-WM262 -shSetdbit 3 140,000 write WM262. ShCGFP 20000 100000 80000 60000 40000 2OOOO O 80 OO 20 140 60 38COO -- WM45 -shSetdbit 3 33000 mis-a-WM45-shCGFP 28OOO 23000 18000 140 160 U.S. Patent Jun. 20, 2017 Sheet 9 of 14 US 9,683,995 B2 Figure C-7 SETDB1 Bound; MCAF/KAP1 not Bound SETDB1. Bound; MCAF/KAP1 Bound SETDB1. Bound (%) SETDBAKAP/MCAF Bound (e) Melf Me3Negative 233 (67) 12S (42) Messive 422 (23) 23 4) e3Positive 1. (6) 28 (9) U.S. Patent Jun. 20, 2017 Sheet 10 of 14 US 9,683,995 B2 O -0.05 & -O,10 III || || || U.S. Patent Jun. 20, 2017 Sheet 11 of 14 US 9,683,995 B2 O -o-minicoopR-SETDBt 6 •r miniCoopR-SETDB (H1224K) 4) or miniCoopR-SETDEst (Cf226A) - miniCoopFEGFP 2 Age (weeks) U.S. Patent Jun. 20, 2017 Sheet 12 of 14 US 9,683,995 B2 WAOI18GLES?6??|87C11HS euoue?aujSOLSeuoue?auuSOLS U.S. Patent Jun. 20, 2017 Sheet 13 of 14 US 9,683,995 B2 Figure 13 Biological sample obtained from the subject Place biological sample in biological sample Container sea Place Container in determination node DEERMINATION SYSTEM 41N to read for a sample, a detectable signal representing the level of SEOB1 expression SORAGE CEVCE COMPARISON WOOE 801-y 90 OO REPORT DAA) REREVED content DiSPAY MODULE: -- 121 9-ya 130 sessessics 3 Krce : rvis sigk as wisgress /140 U.S. Patent Jun. 20, 2017 Sheet 14 of 14 US 9,683,995 B2 Figure 14 START Place biological sample from a subject into determination device 250 Determination system determines the level of expression of SETDB1 mRNA or protein 260 output DATA 1-av Storece output data from determination re module of storage module -- 270 Comparison module Compares output stored data from determination system with a 280 reference sample obtained from (i) reference data from a contro! individual or (ii) reference data from a control population lacking detectable cancer in the tissue from which the biological sample is provided 300 is the eve of SETDB1 expression increased compared to control? Display module indicates Display module indicates subject is likely cancer free subject has cancer or is at in the tissue from which the risk of developing cancer in the tissue from which the sample was provided sample was provided Transmit display data to patient? physician (optional) Program stop 390 US 9,683,995 B2 1. 2 METHOD OF TREATMENT OF SETDB1 Another aspect described herein relates to methods for EXPRESSING CANCER treating cancer comprising administering to a subject an inhibitor of SETDB1. CROSS REFERENCE TO RELATED In one embodiment of this aspect, the Subject is human. APPLICATIONS In another embodiment of this aspect, the method further comprises a step of selecting a subject having cancer. This application is a continuation of U.S. patent applica In another embodiment of this aspect, the selecting step tion Ser. No. 13/147,713 filed Oct. 11, 2011, which is a U.S. comprises comparing the level of SETDB1 in a sample National Stage Entry Application under U.S.C. S371 of the obtained from a subject to a reference, wherein an increase International Application No. PCT/US2010/022994 filed on 10 in SETDB1 level in the sample compared to the reference is Feb. 3, 2010, which designated the U.S., and which claims indicative of cancer in the Subject. the benefit under 35 U.S.C. S 119(e) of U.S. Provisional In another embodiment of this aspect, the level of Application No. 61/149,498, filed Feb. 3, 2009, and U.S. SETDB1 is detected using an antibody-based binding agent Provisional Application No. 61/260,476, filed Nov. 12, 15 which specifically binds to the SETDB1 protein. 2009, the contents of each of which are incorporated herein In another embodiment of this aspect, the cancer is by reference in their entireties. selected from the group consisting of melanoma, thyroid, FIELD OF THE INVENTION pancreas and lung cancer. Also described herein are computer readable storage The field of the invention relates to the diagnosis and/or media having computer readable instructions recorded treatment of cancer. thereon to define software modules including a determina tion system and a comparison module for implementing a BACKGROUND method on a computer for diagnosing a Subject with cancer, the method comprising: (a) storing data derived from a Melanoma is a malignant tumor of melanocytes. Primar 25 biological sample obtained from a subject and which rep ily it is a skin tumor, but it is also seen, though less resents expression data for SETDB1 from a biological frequently, in the melanocytes of the eye (uveal melanoma). sample, (b) comparing with the comparison module the data Even though it represents one of the rarer forms of skin stored on the storage device with reference and/or control cancer, melanoma underlies the majority of skin cancer data, and to provide a retrieved content, and (c) displaying related deaths.
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