Elena Tirrò

Targeting autophagy potentiates tyrosine kinase inhibitor-induced cell death in Philadelphia chromosome-positive cells, including primary CML stem cells.

Imatinib mesylate (IM), a potent inhibitor of the BCR/ABL tyrosine kinase, has become standard first-line therapy for patients with chronic myeloid leukemia (CML), but the frequency of resistance increases in advancing stages of disease. Elimination of BCR/ABL-dependent intracellular signals triggers apoptosis, but it is unclear whether this activates additional cell survival and/or death pathways. We have shown here that IM induces autophagy in CML blast crisis cell lines, CML primary cells, and p210BCR/ABL-expressing myeloid precursor cells. IM-induced autophagy did not involve c-Abl or Bcl-2 activity but was associated with ER stress and was suppressed by depletion of intracellular Ca2+, suggesting it is mechanistically nonoverlapping with IM-induced apoptosis. We further demonstrated that suppression of autophagy using either pharmacological inhibitors or RNA interference of essential autophagy genes enhanced cell death induced by IM in cell lines and primary CML cells. Critically, the combination of a tyrosine kinase inhibitor (TKI), i.e., IM, nilotinib, or dasatinib, with inhibitors of autophagy resulted in near complete elimination of phenotypically and functionally defined CML stem cells. Together, these findings suggest that autophagy inhibitors may enhance the therapeutic effects of TKIs in the treatment of CML.

The Insulin/IGF System in Colorectal Cancer Development and Resistance to Therapy

The insulin/insulin-like growth factor (IGF) system is a major determinant in the pathogenesis and progression of colorectal cancer (CRC). Indeed, several components of this signaling network, including insulin, IGF-1, IGF-2, the IGF-binding proteins, the insulin receptor (IR), the IGF-1 receptor (IGF-1R), and IR substrate proteins 1 and 2 contribute to the transformation of normal colon epithelial cells. Moreover, the insulin/IGF system is also implicated in the development of resistance to both chemotherapeutic drugs and epidermal growth factor receptor targeted agents. The identification of hybrid receptors comprising both the IR and IGF-1R adds further complexity to this signaling network. Thus, a comprehensive understanding of the biological functions performed by each component of the insulin/IGF system is required to design successful drugs for the treatment of CRC patients.

Suppression of Survivin Induced by a BCR-ABL/JAK2/STAT3 Pathway Sensitizes Imatinib-Resistant CML Cells to Different Cytotoxic Drugs

The BCR-ABL oncoprotein of chronic myelogenous leukemia (CML) displays exclusive cytoplasmic localization and constitutive tyrosine kinase activity leading to the activation of different pathways that favor cell proliferation and survival. BCR-ABL induces survivin expression at both the mRNA and protein level, thus inhibiting the apoptotic machinery of CML cells and contributing to the expansion of the leukemic clone. We report that, in human CML cell lines, BCR-ABL–mediated upregulation of survivin involves the JAK2/STAT3 pathway since silencing of either protein caused a consistent reduction in survivin expression. Cell lines unresponsive to imatinib mesylate (IM) because of BCR-ABL gene amplification were not resensitized to the drug after survivin downregulation. However, cells insensitive to IM because of point mutations in the BCR-ABL kinase domain were highly responsive to hydroxyurea (HU) after survivin silencing. To address the possible clinical applications of our results, we used shepherdin, a cell-permeable peptidomimetic compound that downregulates survivin expression by preventing its interaction with Hsp90. Incubation with shepherdin of immortalized cell lines both sensitive and resistant to IM enhanced cell death induced by HU and doxorubicin. Similarly, the combination of shepherdin with first- and second-generation tyrosine kinase inhibitors reduced the colony-forming potential of human progenitors derived from both patients with IM-sensitive and IM-resistant CML. These results suggest that strategies aimed at reducing survivin levels may represent a potential therapeutic option for patients with CML unresponsive to IM. Mol Cancer Ther; 12(6); 1085–98. ©2013 AACR.

BCR-ABL residues interacting with ponatinib are critical to preserve the tumorigenic potential of the oncoprotein

Patients with chronic myeloid leukemia in whom tyrosine kinase inhibitors (TKIs) fail often present mutations in the BCR‐ABL catalytic domain. We noticed a lack of substitutions involving 4 amino acids (E286, M318, I360, and D381) that form hydrogen bonds with ponatinib. We therefore introduced mutations in each of these residues, either preserving or altering their physicochemical properties. We found that E286, M318, I360, and D381 are dispensable for ABL and BCR‐ABL protein stability but are critical for preserving catalytic activity. Indeed, only a “conservative” I360T substitution retained kinase proficiency and transforming potential. Molecular dynamics simulations of BCR‐ABLI360T revealed differences in both helix αC dynamics and protein‐correlated motions, consistent with a modified ATP‐binding pocket. Nevertheless, this mutant remained sensitive to ponatinib, imatinib, and dasatinib. These results suggest that changes in the 4 BCR‐ABL residues described here would be selected against by a lack of kinase activity or by maintained responsiveness to TKIs. Notably, amino acids equivalent to those identified in BCR‐ABL are conserved in 51% of human tyrosine kinases. Hence, these residues may represent an appealing target for the design of pharmacological compounds that would inhibit additional oncogenic tyrosine kinases while avoiding the emergence of resistance due to point mutations.—Buffa, P., Romano, C., Pandini, A., Massimino, M., Tirrò, E., Di Raimondo, F., Manzella, L., Fraternali, F., Vigneri, P. G. BCR‐ABL residues interacting with ponatinib are critical to preserve the tumorigenic potential of the oncoprotein. FASEB J. 28, 1221–1236 (2014). www.fasebj.org

IRF5 is a target of BCR-ABL kinase activity and reduces CML cell proliferation

Interferon regulatory factor 5 (IRF5) modulates the expression of genes controlling cell growth and apoptosis. Previous findings have suggested a lack of IRF5 transcripts in both acute and chronic leukemias. However, to date, IRF5 expression and function have not been investigated in chronic myeloid leukemia (CML). We report that IRF5 is expressed in CML cells, where it interacts with the BCR-ABL kinase that modulates its expression and induces its tyrosine phosphorylation. Tyrosine-phosphorylated IRF5 displayed reduced transcriptional activity that was partially restored by imatinib mesylate (IM). Interestingly, a mutant devoid of a BCR-ABL consensus site (IRF5(Y104F)) still presented significant tyrosine phosphorylation. This finding suggests that the oncoprotein phosphorylates additional tyrosine residues or induces downstream signaling pathways leading to further IRF5 phosphorylation. We also found that ectopic expression of IRF5 decreases the proliferation of CML cell lines by slowing their S-G2 transition, increasing the inhibition of BCR-ABL signaling and enhancing the lethality effect observed after treatment with IM, α-2-interferon and a DNA-damaging agent. Furthermore, IRF5 overexpression successfully reduced the clonogenic ability of CML CD34-positive progenitors before and after exposure to the above-indicated cytotoxic stimuli. Our data identify IRF5 as a downstream target of the BCR-ABL kinase, suggesting that its biological inactivation contributes to leukemic transformation.

New Insights in Thyroid Cancer and p53 Family Proteins

Thyroid cancers are common endocrine malignancies that comprise tumors with different clinical and histological features. Indeed, papillary and follicular thyroid cancers are slow-growing, well-differentiated tumors, whereas anaplastic thyroid cancers are undifferentiated neoplasias that behave much more aggressively. Well-differentiated thyroid carcinomas are efficiently cured by surgery and radioiodine, unlike undifferentiated tumors that fail to uptake radioactive iodine and are usually resistant to chemotherapy. Therefore, novel and more effective therapies for these aggressive neoplasias are urgently needed. Whereas most genetic events underlying the pathogenesis of well-differentiated thyroid cancers have been identified, the molecular mechanisms that generate undifferentiated thyroid carcinomas are still unclear. To date, one of the best-characterized genetic alterations leading to the development of poorly differentiated thyroid tumors is the loss of the p53 tumor suppressor gene. In addition, the existence of a complex network among p53 family members (p63 and p73) and their interactions with other factors that promote thyroid cancer progression has been well documented. In this review, we provide an update on the current knowledge of the role of p53 family proteins in thyroid cancer and their possible use as a therapeutic target for the treatment of the most aggressive variants of this disease.

Concomitant and feasible treatment with dasatinib and the anti- eGFr antibody cetuximab plus radiotherapy in a CMl patient with multiple squamous neoplasias

The advent of biological targeted therapies has changed the prognosis of several hematologic and solid malig-nancies including Chronic Myeloid Leukemia (CML), breast, colo-rectal (CrC) and head and neck squamous cell carcinomas (HNSCC) [1].Dasatinib (DAS) is a multitarget inhibitor of the ABL and SrC tyrosine kinases, currently approved for CML patients resistant or intolerant to imatinib (IM) [2]. Cetuximab (CTX) is an anti-EgFr antibody employed for the treatment of metastatic CrC or, in association with external beam radiation therapy (EBr T), for locally advanced HNSCCs [3,4]. We report the case of a CML patient who developed both benign and malignant squamous lesions during the course of his hematologic disease and was treated concomitantly with DAS and the association of EBrT and CTX.In 1991, a 54-year-old Caucasian male was diag-nosed with Chronic Phase CML (low Sokal risk; BCr-ABL-transcript: e13a2). The patient started α-interferon (9 MUI/daily) obtaining a complete hematological remission (CHr) that he maintained until February 1996 when he was switched to Hydroxyurea (HU) since he lost his CHr. In May 1996, the patient devel -oped a scalp spinocellular epithelioma that was surgi-cally removed. Four years later, still on HU, he was diagnosed with a keratoacanthoma of the left ear that was also excised. In January 2001, he began conventional IM therapy (400 mg/daily), achieving a CHr without any cytogenetic response (Cyr) after 12 months of therapy. He was therefore considered resistant to conventional IM dosage and escalated to 800 mg/daily but had to return to 400 mg because of the repeated occurrence of grade 3/4 thrombocytopenia. In November 2006, he lost his CHr and was placed on DAS (140 mg/daily). At that time, his BCr-ABL/ABL ratio was 106.838

Uncommon long-term survival in a patient with chronic myeloid leukemia

Trials Group. J Clin Oncol 2008;/26:/1871 8. [7] Choi BS, Robins HI. Reversible paclitaxel-induced vocal cord paralysis with later recall with vinorelbine. Cancer Chemother Pharmacol 2008;/61:/345 6. [8] Dodge-Khatami A, Backer CL, Holinger LD, Mavroudis C, Cook KE, Crawford SE. Healing of a free tracheal autograft is enhanced by topical vascular endothelial growth factor in an experimental rabbit model. J Thorac Cardiovasc Surg 2001;/122:/554 61. [9] Walner DL, Heffelfinger SC, Stern Y, Abrams MJ, Miller MA, Cotton RT. Potential role of growth factors and extracellular matrix in wound healing after laryngotracheal reconstruction. Otolaryngol Head Neck Surg 2000;/122:/ 363 6. [10] Schroeder JW, Jr., Rastatter JC, Walner DL. Effect of vascular endothelial growth factor on laryngeal wound healing in rabbits. Otolaryngol Head Neck Surg 2007;/137:/ 465 70. [11] Thibeault SL, Smith ME, Peterson K, Ylitalo-Moller R. Gene expression changes of inflammatory mediators in posterior laryngitis due to laryngopharyngeal reflux and evolution with ppi treatment: A preliminary study. Laryngoscope 2007;/117:/2050 6. [12] Sato K, Hirano M, Nakashima T. Electron microscopic and immunohistochemical investigation of Reinke’s edema. Ann Otol Rhinol Laryngol 1999;/108:/1068 72. [13] Tse GM, Chan AW, Yu KH, King AD, Wong KT, Chen GG, et al. Strong immunohistochemical expression of vascular endothelial growth factor predicts overall survival in head and neck squamous cell carcinoma. Ann Surg Oncol 2007;/14:/3558 65.

Non ABL-directed inhibitors as alternative treatment strategies for chronic myeloid leukemia

The introduction of ABL Tyrosine Kinase Inhibitors (TKIs) has significantly improved the outcome of Chronic Myeloid Leukemia (CML) patients that, in large part, achieve satisfactory hematological, cytogenetic and molecular remissions. However, approximately 15–20% fail to obtain optimal responses according to the current European Leukemia Network recommendation because of drug intolerance or resistance.Moreover, a plethora of evidence suggests that Leukemic Stem Cells (LSCs) show BCR-ABL1-independent survival. Hence, they are unresponsive to TKIs, leading to disease relapse if pharmacological treatment is discontinued.All together, these biological events generate a subpopulation of CML patients in need of alternative therapeutic strategies to overcome TKI resistance or to eradicate LSCs in order to allow cure of the disease.In this review we update the role of “non ABL-directed inhibitors” targeting signaling pathways downstream of the BCR-ABL1 oncoprotein and describe immunological approaches activating specific T cell responses against CML cells.

Roles of Interferon Regulatory Factors in Chronic Myeloid Leukemia

The Interferon Regulatory Factor (IRF) family consists of multiple transcription factors involved in the regulation of a variety of biological processes. Originally identified as transcriptional regulators of the type I interferon system, IRFs play a pivotal role in adaptive immunity, cell growth, differentiation and tumorigenesis. Hence, understanding IRF biology has important implications in the host response to cancer development and progression. Many lines of evidence suggest that different IRFs are involved in the pathogenesis of Chronic Myeloid Leukemia (CML), a myeloproliferative disorder caused by the BCR-ABL oncoprotein. BCR-ABL displays constitutive tyrosine kinase activity that favors cell proliferation, inhibits apoptosis and allows cell survival even in the absence of proper adhesion to the extracellular matrix. Different BCR-ABL tyrosine kinase inhibitors are currently available for CML treatment. These drugs are able to generate eight year CML-specific overall survival rates >90%, only a minority of patients will achieve molecular responses compatible with drug discontinuation. Thus, there is an unmet need for additional therapeutic targets that may lead to the cure of most patients diagnosed with CML. A growing body of evidence has suggested a role for both IRF4 and IRF8 in the pathogenesis of CML. Furthermore, IRF1 is consistently deleted at one or both alleles in patients with leukemia and myelodysplasia. Finally, we have recently demonstrated that IRF5 is a target of BCR-ABL kinase activity and reduces CML cell proliferation. In this article, we provide an update on the current knowledge of the role of the IRFs in CML.