Modelling Myc inhibition as a cancer therapy

Abstract

Myc is a pleiotropic basic helix-loop-helix leucine zipper transcription factor that coordinates expression of the diverse intracellular and extracellular programs that together are necessary for growth and expansion of somatic cells. In principle, this makes inhibition of Myc an attractive pharmacological approach for treating diverse types of cancer. However, enthusiasm has been muted by lack of direct evidence that Myc inhibition would be therapeutically efficacious, concerns that it would induce serious side effects by inhibiting proliferation of normal tissues, and practical difficulties in designing Myc inhibitory drugs. We have modelled genetically both the therapeutic impact and the side effects of systemic Myc inhibition in a preclinical mouse model of Ras-induced lung adenocarcinoma by reversible, systemic expression of a dominant-interfering Myc mutant. We show that Myc inhibition triggers rapid regression of incipient and established lung tumours, defining an unexpected role for endogenous Myc function in the maintenance of Ras-dependent tumours in vivo. Systemic Myc inhibition also exerts profound effects on normal regenerating tissues. However, these effects are well tolerated over extended periods and rapidly and completely reversible. Our data demonstrate the feasibility of targeting Myc, a common downstream conduit for many oncogenic signals, as an effective, efficient and tumour-specific cancer therapy.

Figure 1. Endogenous Myc function is required for formation and maintenance of early-stage Kras-induced lung hyperplasias/adenomas

a, Representative haematoxylin-and-eosin-stained sections of lungs from CMVrtTA single transgenic mice (CMVrtTA+doxycycline), untreated Kras;TRE-Omomyc;CMVrtTA triple transgenic mice (Kras;TRE-Omomyc; CMVrtTA − doxycycline) and doxycycline-treated LSL-Kras;TRE-Omomyc;CMVrtTA triple transgenic mice (Kras;TRE-Omomyc;CMVrtTA+doxycycline), 4 weeks after infection with adenoviral Cre. Hyperplastic lesions are indicated by black arrowheads. b, Graphical representation of total BADJ cells scored as proliferating (Ki67-positive). Error bars represent standard deviation derived from approximately 100 BADJs per mouse. At least three mice were used per series. c, Myc inhibition triggers regression of early-stage lung adenomas. Haematoxylin and eosin staining of lungs from mice treated or not with doxycycline for 1 week, starting 6 weeks after Cre-recombinase-expressing adenovirus infection, is shown. A small adenoma is indicated by black arrowheads. d, TUNEL staining reveals positive cells in Omomyc and Ras co-expressing samples (Kras;TRE-Omomyc;CMVrtTA+doxycycline), but not in untreated or single transgenic controls (Kras;TRE-Omomyc;CMVrtTA −doxycycline and CMVrtTA +doxycycline). Higher magnification of positive cells is shown in the insert on the right.

Figure 2. Myc inhibition elicits regression of established lung tumours

a, Haematoxylin-and-eosin-stained lungs from control CMVrtTA single transgenic mice (CMVrtTA+doxycycline), Kras^G12D-expressing mice (Kras;TRE-Omomyc;CMVrtTA − doxycycline) and 3- or 6-day-treated Kras^G12D and Omomyc co-expressing mice (Kras;TRE-Omomyc;CMVrtTA +doxycycline) 18 weeks after infection with adenovirus expressing Cre recombinase. An example of a frank tumour is shown from a mouse expressing Kras^G12D only (Kras;TRE-Omomyc;CMVrtTA −doxycycline). b, BrdU staining shows a considerable reduction in BrdU-positive cells in Kras^G12D and Omomyc co-expressing mice (Kras;TRE-Omomyc;CMVrtTA +doxycycline) compared with tissues from mice expressing Kras^G12D only (Kras;TRE-Omomyc;CMVrtTA −doxycycline). c, TUNEL staining indicates the presence of apoptotic cells in Omomyc and Kras^G12D co-expressing sections (Kras;TRE-Omomyc;CMVrtTA + doxycycline), but not in untreated or single transgenic control tissues (Kras;TRE-Omomyc; CMVrtTA −doxycycline and CMVrtTA +doxycycline). d, Haematoxylin and eosin staining of lung tissue from mice treated for 4 weeks with doxycycline shows clearance of tumour lesions as a consequence of Omomyc expression (compare Kras;TRE-Omomyc; CMVrtTA +doxycycline with Kras;TRE-Omomyc;CMVrtTA −doxycycline).

Figure 3. Inhibition of endogenous Myc suppresses proliferation in skin, testis and GI tract

a, Anti-Ki67-stained sections of epidermis from 4-week doxycycline-treated TRE-Omomyc;CMVrtTA mice and CMVrtTA single transgenic controls. b, Anti-Ki67-stained sections of testis from 4-week doxycycline-treated TRE-Omomyc;CMVrtTA mice show reduced proliferation in seminiferous tubules of doxycycline-treated TRE-Omomyc; CMVrtTA mice compared with Omomyc-negative controls. c, Haematoxylin-and-eosin-stained small intestine sections show blunted villi in TRE-Omomyc;CMVrtTA mice treated for 4 weeks with doxycycline (TRE-Omomyc;CMVrtTA +doxycycline) compared with intestine from untreated TRE-Omomyc;CMVrtTA and doxycycline-treated CMVrtTA single transgenic (CMVrtTA+doxycycline) controls.

Figure 4. The degenerative phenotypes induced by systemic Myc inhibition are rapidly and completely reversible on restoration of Myc function

Myc transactivation function was blocked for 4 weeks by sustained Omomyc expression. Doxycycline was then withdrawn for 1 week. a, Ki67 staining indicates rapid recovery of cell proliferation in skin. b, Rapid recovery of spermatogenesis in seminiferous tubules. Haematoxylin-and-eosin-stained sections from Omomyc-expressing testis (left panel) and the same tissue 1 week after discontinuing Omomyc expression (right panel) are shown. c, Rapid recovery of intestinal villus architecture in TRE-Omomyc;CMVrtTA mice after restoration of Myc function. After 4 weeks of sustained Omomyc expression, doxycycline treatment was discontinued and cohorts of mice killed each day for 5 days after doxycycline withdrawal. Representative haematoxylin-and-eosin-stained sections are shown for each time point.