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Acute lymphoblastic leukemia (ALL) is an aggressive blood cancer that mainly affects children. Relapse rates are high and toxic chemotherapies that block DNA replication and induce DNA damage lead to health problems later in life, underlining the need for improved therapies. MYC is a transcription factor that is hyperactive in a large proportion of cancers including leukemia but is difficult to target in therapy. We show that ablation of the function of the BTB/POZ domain factor Zbtb17 (Miz-1), an important cofactor of c-Myc, significantly delayed T- and B-ALL/lymphoma in mice and interfered with the oncogenic transcriptional activity of c-Myc. Leukemic cells that still emerged in this system activated DNA replication pathways that could be targeted by current chemotherapeutic drugs such as cytarabine. Acute ablation of the Miz-1 POZ domain enhanced the effect of cytarabine treatment. The combined treatment was effective in both Eμ-Myc and Notch ICN-driven leukemia models and prolonged survival of tumor-bearing animals by accelerating apoptosis of leukemic cells. These observations suggest that targeting MIZ-1 could render current ALL chemotherapies more effective, with a better outcome for patients. SIGNIFICANCE: Ablation of the POZ domain of Miz-1 perturbs its interaction with c-MYC and delays the generation of T- and B-cell leukemias and lymphomas., (©2019 American Association for Cancer Research.)

To be effective, the adaptive immune response requires a large repertoire of antigen receptors, which are generated through V(D)J recombination in lymphoid precursors. These precursors must be protected from DNA damage-induced cell death, however, because V(D)J recombination generates double-strand breaks and may activate p53. Here we show that the BTB/POZ domain protein Miz-1 restricts p53-dependent induction of apoptosis in both pro-B and DN3a pre-T cells that actively rearrange antigen receptor genes. Miz-1 exerts this function by directly activating the gene for ribosomal protein L22 (Rpl22), which binds to p53 mRNA and negatively regulates its translation. This mechanism limits p53 expression levels and thus contains its apoptosis-inducing functions in lymphocytes, precisely at differentiation stages in which V(D)J recombination occurs.

Miz-1 is a Broad-complex, Tramtrack and Bric-à-brac/pox virus zinc finger domain (BTB/POZ)-containing protein expressed in lymphoid precursors that can activate or repress transcription. We report in this article that mice expressing a nonfunctional Miz-1 protein lacking the BTB/POZ domain (Miz-1(ΔPOZ)) have a severe differentiation block at the pre-T cell "β-selection" checkpoint, evident by a drastic reduction of CD4(-)CD8(-) double-negative-3 (DN3) and DN4 cell numbers. T cell-specific genes including Rag-1, Rag-2, CD3ε, pTα, and TCRβ are expressed in Miz-1-deficient cells and V(D)J recombination is intact, but few DN3/DN4 cells express a surface pre-TCR. Miz-1-deficient DN3 cells are highly apoptotic and do not divide, which is consistent with enhanced expression of p53 target genes such as Cdkn1a, PUMA, and Noxa. However, neither coexpression of the antiapoptotic protein Bcl2 nor the deletion of p21(CIP1) nor the combination of both relieved Miz-1-deficient DN3/DN4 cells from their differentiation block. Only the coexpression of rearranged TCRαβ and Bcl2 fully rescued Miz-1-deficient DN3/DN4 cell numbers and enabled them to differentiate into DN4TCRβ(+) and double-positive cells. We propose that Miz-1 is a critical factor for the β-selection checkpoint and is required for both the regulation of p53 target genes and proper expression of the pre-TCR to support the proliferative burst of DN3 cells during T cell development.

B cell development requires the coordinated action of transcription factors and cytokines, in particular interleukin-7 (IL-7). We report that mice lacking the POZ (Poxvirus and zinc finger) domain of the transcription factor Miz-1 (Zbtb17(ΔPOZ/ΔPOZ)) almost entirely lacked follicular B cells, as shown by the fact that their progenitors failed to activate the Jak-Stat5 pathway and to upregulate the antiapoptotic gene Bcl2 upon IL-7 stimulation. We show that Miz-1 exerted a dual role in the interleukin-7 receptor (IL-7R) pathway by directly repressing the Janus kinase (Jak) inhibitor suppressor of cytokine signaling 1 (Socs1) and by activating Bcl2 expression. Zbtb17(ΔPOZ/ΔPOZ) (Miz-1-deficient) B cell progenitors had low expression of early B cell genes as transcription factor 3 (Tcf3) and early B cell factor 1 (Ebf1) and showed a propensity for apoptosis. Only the combined re-expression of Bcl2 and Ebf1 could reconstitute the ability of Miz-1-deficient precursors to develop into CD19(+) B cells.

The Myc protein suppresses the transcription of several cyclin-dependent kinase inhibitors (CKIs) via binding to Miz1; whether this interaction is important for Myc's ability to induce or maintain tumorigenesis is not known. Here we show that the oncogenic potential of a point mutant of Myc (MycV394D) that is selectively deficient in binding to Miz1 is greatly attenuated. Binding of Myc to Miz1 is continuously required to repress CKI expression and inhibit accumulation of trimethylated histone H3 at Lys 9 (H3K9triMe), a hallmark of cellular senescence, in T-cell lymphomas. Lymphomas that arise express high amounts of transforming growth factor beta-2 (TGFbeta-2) and TGFbeta-3. Upon Myc suppression, TGFbeta signaling is required to induce CKI expression and cellular senescence and suppress tumor recurrence. Binding of Myc to Miz1 is required to antagonize growth suppression and induction of senescence by TGFbeta. We demonstrate that, since lymphomas express high levels of TGFbeta, they are poised to elicit an autocrine program of senescence upon Myc inactivation, demonstrating that TGFbeta is a key factor that establishes oncogene addiction of T-cell lymphomas.

Previous work has implicated the Myc-binding transcription factor Miz1 in the control of keratinocyte proliferation and in the cellular response to TGFbeta. Miz1 is expressed in basal keratinocytes of the interfollicular epidermis and in hair follicles. Here we have conditionally knocked out the POZ/BTB transactivation domain of Miz1 in keratinocytes using a keratin 14 (K14)-Cre mouse deleter strain. K14Cre(+)/Miz1(lox/lox) mice have rough fur as a result of altered hair follicle orientation, irregular hair pigmentation and disturbed hair fiber structure. A regional thickening of the epidermis at the hair funnel orifice was accompanied by suprabasal proliferation, indicating a delayed exit of keratinocytes from the cell cycle. In addition, the catagen of the hair cycle was delayed in K14Cre(+)/Miz1(lox/lox) mice and intrafollicular keratinocyte proliferation was increased. In aged K14Cre(+)/Miz1(lox/lox) animals, the number of hair follicles remained unchanged but the number of visible hairs, especially of zigzag hairs, was reduced and a pigmentary incontinence into the dermis developed. Our data show that Miz1 is involved in controlling proliferation and differentiation in hair follicles and in hair fiber morphogenesis.