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The coordination of transcription through enhancers and promoters is a fundamental process that has a central role in development and disease 1–3. Convergent transcription, i.e., the collision of sense and antisense transcription, is ubiquitous in mammalian genomes 4–8 and believed to diminish RNA expression 9,10. Recently, antisense transcription downstream of promoters was found to be surprisingly prevalent 11–13. However, functional characteristics of promoters with convergent transcription are unknown. Here, we show that convergent transcription marks an unexpectedly cooperative promoter constellation we call super-promoters. By assessing transcriptional dynamic systems, we identified strongly cooperative constituent promoters that are connected through a distinct chromatin structure. Within these super-promoter constellations transcription factors can regulate both constituting promoters by binding to only one of them. Super-promoters comprise about a fourth of all active transcript starts and initiate 5’-overlapping antisense RNAs, an RNA class previously believed to be rare 4. Visualization of nascent RNA molecules provides evidence for convergent co-transcription at these loci. Our results demonstrate that super-promoters substantially expand the cis-regulatory repertoire and reveal limitations of the standing model of transcription interference. Together, our study revises the promoter architecture of many genes and calls for adjusting the promoter concept.

Recurrently mutated in lymphoid neoplasms, the transcription factor RFX7 is emerging as a tumor suppressor. Previous reports suggested that RFX7 may also have a role in neurological and metabolic disorders. We recently reported that RFX7 responds to p53 signaling and cellular stress. Furthermore, we found RFX7 target genes to be dysregulated in numerous cancer types also beyond the hematological system. However, our understanding of RFX7’s target gene network and its role in health and disease remains limited. Here, we generated RFX7 knock-out cells and employed a multi-omics approach integrating transcriptome, cistrome, and proteome data to obtain a more comprehensive picture of RFX7 targets. We identify novel target genes linked to RFX7’s tumor suppressor function and underscoring its potential role in neurological disorders. Importantly, our data reveal RFX7 as a mechanistic link that enables the activation of these genes in response to p53 signaling.

In recent years the tumor suppressor p53 has been increasingly recognized as a potent regulator of the cell metabolism and for its ability to inhibit the critical pro-survival kinases AKT and mTOR. The mechanisms through which p53 controls AKT and mTOR, however, are largely unclear. Here, we demonstrate that p53 activates the metabolic regulator DDIT4 indirectly through the regulatory factor X 7 (RFX7). We provide evidence that DDIT4 is required for p53 to inhibit mTOR complex 2 (mTORC2)-dependent AKT activation. Most strikingly, we also find that the DDIT4 regulator RFX7 is required for p53-mediated inhibition of mTORC1 and AKT. Our results suggest that AMPK activation plays no role and p53-mediated AKT inhibition is not critical for p53-mediated mTORC1 inhibition. Moreover, using recently developed physiological cell culture media we uncover that basal p53 and RFX7 activity can play a critical role in restricting mTORC1 activity under physiological nutrient conditions, and we propose a nutrient-dependent model for p53-RFX7-mediated mTORC1 inhibition. These results establish RFX7 and its downstream target DDIT4 as essential effectors in metabolic control elicited by p53.

Despite its prominence, the mechanisms through which the tumor suppressor p53 regulates most genes remain unclear. Recently, the regulatory factor X 7 (RFX7) emerged as a suppressor of lymphoid neoplasms, but its regulation and target genes mediating tumor suppression remain unknown. Here, we identify a novel p53-RFX7 signaling axis. Integrative analysis of the RFX7 DNA binding landscape and the RFX7-regulated transcriptome in three distinct cell systems reveals that RFX7 directly controls multiple established tumor suppressors, including PDCD4, PIK3IP1, MXD4, and PNRC1, across cell types and is the missing link for their activation in response to p53 and stress. RFX7 target gene expression correlates with cell differentiation and better prognosis in numerous cancer types. Interestingly, we find that RFX7 sensitizes cells to Doxorubicin by promoting apoptosis. Together, our work establishes RFX7’s role as a ubiquitous regulator of cell growth and fate determination and a key node in the p53 transcriptional program., Graphical Abstract Graphical Abstractp53 induction by MDM2-inhibition, DNA-damage, or ribosomal stress activates the transcription factor RFX7. In turn, RFX7 up-regulates multiple tumor suppressor genes.

Introduction: Acute lymphoblastic leukemia (ALL) represents approx. 5% of all newly diagnosed leukemias in patients between 55 and 70 years of age. Despite recent advances especially in younger patients, the prognosis of elderly patients with ALL remains dismal, even after moderately intensive chemotherapy. Incorporation of antibody-based therapies in the first line induction therapy bears the potential of a significant increased response rate and survival with the reduction of treatment toxicity. Methods: This open label phase II study of the German Multicenter Study Group on Adult Acute Lymphoblastic Leukemia was conducted at 13 centers. Patients aged >55 years with newly diagnosed Ph/BCR-ABL negative acute B-precursor ALL were eligible. Leukemic blasts had CD22 surface expression of at least 20%. The 1st induction cycle consisted of InO 0.8mg/m2 on day1 and 0.5mg/m2 on d8 and d15 together with dexamethasone 10 mg/m2 (day7-8, day14-17) and one intrathecal injection (i.th.) of methotrexate (MTX), cytarabine (AraC) and dexamethasone (Dex). The 2nd and 3rd induction cycle consisted of InO 0.5mg/m2 on day 1, 8 and 15 plus i.th. MTX/AraC/Dex. Patients achieving a complete remission (CR) were offered to receive 5 conventional consolidation therapies (3 x ID-MTX/asparaginase; 2 x ID-AraC) and one reinduction therapy (idarubicine/ AraC/ cyclophosphamide / Dex.) in combination with rituximab (for CD20+ ALL), followed by a maintenance therapy with 6-MP/MTX. The primary endpoint was event free survival (EFS) at 12-months follow-up. An event was defined as any of the following: persisting bone marrow blasts after 2 cycles of InO, relapse or death. An event rate of ≤40% at 12 months follow-up was considered to qualify the experimental treatment as very promising for additional testing. Under the assumption of one-sided type I error of 5% and 80% power, 42 evaluable patients were needed for primary endpoint analysis (registered with ClinicalTrials.gov, identifier: NCT03460522). Results: As of July 2021, 45 patients were included, with induction results available for 43 patients. Two patients were excluded per protocol from further analysis, as they received only one induction: 1 prolonged toxicity (CR after induction), 1 withdrawn consent. Median age at initial diagnosis was 64 years (range 56-80 years). 38 patients were diagnosed with a common- and 5 with a pro-B ALL. Median CD22 expression on leukemic blasts was 69% (range 21-99%). Due to suspected therapy related liver toxicities, a single patient received only 2 induction cycles (complete remission after 1st induction). All other patients completed 3 cycles of induction therapy and achieved complete remission (CR/CRi), mainly after the 1st induction. Results of minimal residual disease (MRD), measured by real-time quantitative PCR, are available for all 43 patients, with 23/43 (53%) and 31/42 (74%) patients being MRD negative after 2nd and 3rd induction therapy, respectively. Four patients with MRD failure (≥10 -4) after the 3rd induction therapy are still in CR (2 were treated with conventional chemotherapy per this protocol, one proceeded to allogeneic stem cell transplantation (aSCT), one received blinatumomab). MRD below the threshold of 10 -4 was detected in 15 patients after the 2nd induction (no relapse occurred so far). 8 of these 15 patients were MRD negative after the 3rd induction. So far, 7 events have been reported (during year 1: 3 deaths in CR and 1 relapsed ALL; during year 2: 1 death in CR and 2 patients with relapse). With a median follow-up of 422 days, the probability of OS at 1 and 2 years is 91% (95% C.I. 80-100%) and 77% (95% C.I. 57-96%), respectively. 4 patients received an aSCT in 1st CR, 1 patient in 2nd CR. Regarding adverse events (AEs) during induction cycles 1, 2 and 3, most common AEs ≥CTC grade 3 reported were leukocytopenia (in 60%, 12% and 3% of all cases, respectively), anemia (28%, 2%, 0%), thrombocytopenia (35%, 7%, 3%) and elevation of liver enzymes (14%, 5%, 0%). So far, one patient has been reported with suspected veno occlusive disease (after 2nd induction therapy). Conclusion: Three cycles of InO as induction therapy for the treatment of elderly patients with acute B-cell ALL results in very high remission rates with MRD-response in up to 90% (MRD negativity or MRD-levels Figure 1 Figure 1. Disclosures Stelljes: Pfizer: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Medac: Speakers Bureau; MSD: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Celgene/BMS: Consultancy, Speakers Bureau; Kite/Gilead: Consultancy, Speakers Bureau. Wäsch: Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Pfizer: Consultancy; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Novartis: Consultancy; BMS/Celgene: Consultancy; Gilead: Consultancy. Nachtkamp: Jazz: Honoraria; Bsh medical: Honoraria; Celgene: Other: Travel Support. Haenel: Jazz: Consultancy, Honoraria; GSK: Consultancy; Bayer Vital: Honoraria; Takeda: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy; Celgene: Consultancy, Honoraria. Berdel: Philogen S.p.A.: Consultancy, Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees. Goekbuget: Amgen: Consultancy, Other: Invited talks for company sponsored symposia (with honoraria); Research funding for institution; Astra Zeneca: Other: Invited talk for company sponsored symposia (with honor); Erytech: Consultancy; Cellestia: Consultancy; Incyte: Other: Research funding for Institution; Jazz Pharmaceuticals: Other: Research funding for institution; Novartis: Consultancy, Other: Research funding for Institution; Gilead/Kite: Consultancy; Morphosys: Consultancy; Pfizer: Consultancy, Other: Research funding for institution; Servier: Consultancy, Other; Abbvie: Other. OffLabel Disclosure: Inotuzumab Ozogamicin

Introduction: Acute lymphoblastic leukemia (ALL) represents around 5% of all newly diagnosed leukemia in patients between 55 and 70 years of age. Despite recent advances especially in younger patients, the prognosis of older patients with ALL remains dismal, even after moderately intensive chemotherapy. Due to increasing toxicity and infection rates in older patients, dose intensification of induction treatment often is no justifiable option. Consequently, new treatment options are needed to improve the survival of older ALL patients. Methods: This open label phase II study of the German Multicenter Study Group on Adult Acute Lymphoblastic Leukemia (GMALL) is currently activated in 14 centers in Germany. Patients aged >55 years with newly diagnosed acute B lymphoblastic leukemia, with the exception of Philadelphia-chromosome or BCR-ABL positive ALL or Burkitt's or mixed phenotype acute leukemia, are eligible. Leukemic blasts must have CD22 surface expression of at least 20%. No previous ALL-specific treatment, with the exception of corticosteroids and/or single dose vincristine and/or up to 3 doses of cyclophosphamide (cycloph.) plus standard prephase treatment are allowed. The 1st induction cycle consists of inotuzumab ozogamicin (InO) 0.8 mg/m2 on day1 and 0.5 mg/m2 on days 8 and 15 together with dexamethasone 10 mg/m2 (days 7-8, days 14-17) and 1 intrathecal injection of methotrexate (MTX), cytarabine (AraC) and dexamethasone (Dexa). The 2nd and 3rd induction cycle consist of InO 0.5 mg/m2 on days 1, 8 and 15 plus intrathecal injection of MTX/AraC/Dexa. Response evaluation is scheduled after each cycle. Patients achieving a complete remission are offered to receive 5 conventional consolidation therapies (3 x ID-MTX/asparaginase; 2 x ID-AraC) and one reinduction therapy (idarubicine/AraC/cycloph./Dexa) in combination with rituximab (for CD20+ ALL), followed by a maintenance therapy with 6-mercaptopurine/MTX. The primary endpoint is is event free survival (EFS) at 12-months follow-up. An event is any of the following: persisting bone marrow blasts after 2 cycles of InO, relapse or death. An event rate of ≤40% at 12 months follow-up is considered to qualify the experimental treatment as very promising for additional testing. Under the assumption of one-sided type I error of 5% and 80% power, 42 evaluable patients were needed for primary endpoint analysis. The INITIAL-1 trial is registered with ClinicalTrials.gov, identifier: NCT03460522. Results: As of July 2020, 31 patients have been included, with induction results available for 29 patients. Median age at initial diagnosis was 64 years (range 56-80 years). Twenty-five patients were diagnosed with a common- and 4 with a pro-B lymphoblastic leukemia. Median CD22 expression on leukemic blasts was 70% (range 21-99%). Due to suspected therapy related liver toxicities, 1 patient received 1 induction cycle and 1 patient 2 induction cycles (both were in remission after the 1st induction). All other patients completed induction therapy and achieved complete remission (CR/CRi) mainly after the 1st induction. Results of minimal residual disease (MRD) measured by PCR are available for 23 patients, with 17 being MRD-negative after induction. So far, 4 events have been reported (2 deaths in remission and 1 relapsed ALL in the 1st year of treatment; one relapsed disease in the 2nd year). With a median follow-up of 242 days, the probability of OS at 1 year is 82.4 %. Two patients received an allogeneic stem cell transplantation in ongoing 1st remission. With regard to adverse events (AEs) during induction therapy 1, 2 and 3, most common AEs ≥CTC 3 reported were leukocytopenia (in 64%, 33% and 13% of all cases, respectively), anemia (54%, 28%, 13%), thrombocytopenia (68%, 17%, 26%) and elevation of liver enzymes (31%, 22%, 20%). Conclusion: Replacement of conventional induction chemotherapy by InO is feasible, results in promising remission rates, and may reduce the risk of early morbidity and lethality, particular in older patients with acute B lymphoblastic leukemia. Disclosures Stelljes: Amgen: Consultancy, Speakers Bureau; Pfizer: Consultancy, Honoraria, Research Funding, Speakers Bureau. Wäsch:Pfizer: Consultancy; Amgen: Consultancy; Janssen: Consultancy. Haenel:Amgen, Novartis, Roche, Celgene, Takeda, Bayer: Honoraria. Lenz:Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy; AstraZeneca: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Agios: Research Funding; AQUINOX: Research Funding; Novartis: Consultancy; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Verastem: Research Funding; Morphosys: Consultancy, Honoraria, Research Funding. Brüggemann:Affimed: Research Funding; Regeneron: Research Funding; Celgene: Consultancy; Roche: Consultancy; Incyte: Consultancy; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Goekbuget:Gilead: Consultancy; Kite: Consultancy; Servier: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Erytech: Consultancy; Amgen: Consultancy, Research Funding. Alakel:Pfizer: Consultancy. OffLabel Disclosure: Inotuzumab ozogamicin for induction therapy (1st line therapy)